Endothelin single nucleotide polymorphisms and methods of predicting β-adrenergic receptor targeting agent efficacy

ABSTRACT

The present invention concerns the use of methods for evaluating β-adrenergic receptor targeting agent treatment for a patient, particularly one with a heart condition. In general, the disclosed methods entail determining the presence or absence of one or more polymorphisms in an endothelin gene system member. Based on the results of this determination, a β-adrenergic receptor targeting agent may be prescribed, administered or a treatment regimen altered, including the administration of a β-blocker. Accordingly, methods of treatment are also described.

This application is a national phase application under 35 U.S.C. §371 ofInternational Application No. PCT/US2008/071342 filed Jul. 28, 2008which claims priority to U.S. Provisional Application No. 60/952,441filed Jul. 27, 2007, which is incorporated herein by reference in itsentirety.

This invention was made with government support under grant numbersNIH-1K23HI67915-01A1, HL69071-01 and GM062628-05 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to pharmacogenetics and cardiology. Morespecifically, the present invention relates, in part, to methods ofpredicting the efficacy of β-adrenergic receptor targeting agenttreatment in a patient with a cardiovascular condition based on thepatient's genotype of certain polymorphisms in endothelin gene systemmembers.

2. Description of Related Art

According to the American Heart Association (AHA), about 79 millionAmericans have some form of cardiovascular disease, which can includehigh blood pressure, coronary heart disease (heart attack and chestpain), cardiomyopathy, stroke, birth defects of the heart and bloodvessels, and congestive heart failure, and close to a million die fromsuch conditions every year. The annual report of the AHA further statesthat cardiovascular disease kills more Americans than the next sevencauses of death combined, including cancer. Heart disease accounted for40% of all deaths in the U.S. in 1999, and mortality from heart failureis approximately 50% within 5 years.

In the United States alone there are approximately six million people,about 1.5% of the population, with chronic heart failure (“HF”), androughly 550,000 new patients are diagnosed each year. Medical therapyhas made progress in treating HF, but morbidity and mortality remainhigh (Mann et al., 2005). The current standard of care in HF involvesthe use of inhibitors (ACE inhibitors, ARBs, and/or aldosterone receptorantagonists) of the renin-angiotensin-aldosterone system (RAAS), andβ-blockers, which competitively inhibit β-adrenergic receptors oncardiac myocytes. β-blocker therapy is standard treatment for systolicheart failure and reduces allcause mortality by 34-65% (CIBIS-IIInvestigators, 1999; Packer et al., 1996; Packer et al., 2001). Thesedata are in agreement with other large drug trials where only between25-60% of patients benefit from exposure to medication as compared toplacebo (Wilkinson, 2005; Evans and Relling, 2004; Weinshilboum, 2003).

Even though most β-blocker trials in heart failure have shown groupbeneficial effects, there is substantial interindividual variability inoutcome that is not explained by baseline clinical characteristics(CIBIS-II Investigators, 1999). Interindividual variability in theresponse to pharmacologic therapy is recognized with virtually alldrugs. In circumstances such as the treatment of chronic heart failurewith β-blockers—where morbidity and mortality are high, the titrationalgorithm is complex, the interindividual variability is substantial,and additional treatment options exist—assessing the likelihood of afavorable (or adverse) long-term response to drug therapy can have asignificant impact on decision making.

Another cardiovascular disease, cardiomyopathy, is a disease of theheart muscle. This form of cardiovascular disease is often distinctive,both in general symptoms and in patterns of blood flow, thusfacilitating diagnosis. Increasing recognition of this disease, alongwith improved diagnostic techniques, have shown that cardiomyopathy is amajor cause of morbidity and mortality. In some areas of the world itmay account for as many as 30% of all deaths due to heart disease.

Several types of cardiomyopathy are known, including ischemic, dilated,hypertrophic, restrictive and idiopathic (and combinations thereof). Theprognosis for all types of these diseases is often poor. For example,the survival rate of dilated cardiomyopathy for five years is typically50 to 60%. Treatment of cardiomyopathy involves restricted activity,stress avoidance, treatment with β-blockers, prophylactic antibiotictherapy, use of anti-coagulants, calcium channel blockers, surgery andcardiac transplantation. With this variety of treatments available, itwould be helpful to identify which patients would benefit the most fromone treatment as compared to another. For example, dobutamine stresscardiovascular magnetic resonance has been employed to predict whetheror not β-blocker treatment of patients exhibiting severe ischemiccardiomyopathy will be beneficial (Kaandorp 2005). However, the smallnumber of patients tested limits the significance and clinicalapplicability of these findings. Other means of predicting whichpatients will benefit from β-blocker treatment are also needed.

SUMMARY OF THE INVENTION

The present invention provides methods for individualized therapy basedon the identification of polymorphisms in endothelin gene system membersthat affect an individual's response to β-adrenergic receptor targetingagents, such as β-blockers. In certain embodiments, the presentinvention concerns individualized therapy for heart failure.

The invention is based, in part, on the determination by the inventorsthat certain single nucleotide polymorphisms (SNPs) in an endothelin(EDN) gene system member of a patient suffering from a medical conditionrender that patient amenable to treatment with a β-adrenergic receptortargeting agent. For example, as discussed in more detail below, allelesrepresented by two EDN1 SNPs, Lys198Asn and G/A (Intervening sequence(IVS)-4)), commonly appearing in one of two complementary haplotypes,showed a strong pharmacogenetic interaction with bucindolol therapy ontime to first heart failure hospitalization or all cause mortality.These results appear to represent the first study to identify ahaplotype associated with clinical response to a β-blocker in dilatedcardiomyopathy. The findings discussed herein demonstrate that certainEDN-related genetic predictors of outcome may identify a largesubpopulation of patients who may benefit from β-adrenergic receptortargeting agent treatment.

Accordingly, in one general aspect, the present invention contemplates amethod for evaluating β-adrenergic receptor targeting agent treatment ofa patient comprising determining the presence or absence of at least onepolymorphism in an endothelin gene system member of the patient, whereinthe information is predictive of β-adrenergic receptor targeting agentefficacy in the patient. Methods of the present invention also includeassessing whether to prescribe or administer a β-adrenergic receptortargeting agent to a patient with a medical condition, such ascardiovascular disease, comprising obtaining information from thepatient regarding his/her polymorphisms in endothelin gene systemmembers and/or their encoded gene products that affect a response to aβ-adrenergic receptor targeting agent. In certain embodiments, knowledgeabout which polymorphism a patient has at (i) nucleotide position +356in intron 4 of EDN1 (rs2071942) or (ii) nucleotide position +61 in exon5 of EDN1 (rs5370) encoding a polymorphism at amino acid 198 of EDN1protein provides the basis for assessing whether to administer orprescribe a β-adrenergic receptor targeting agent to the patient. Theterms “rs2071942” and “rs5370” refer to the Reference SNP (rs)identification numbers used in standard nomenclature by the NationalCenter for Biotechnology Information (NCBI).

It is generally understood that polymorphisms occur in the context ofgenes; however, in the case of polymorphisms that affect the encodedgene product, an alteration in that gene product may also be referred toas a polymorphism. For example, in certain embodiments, at least onepolymorphism is at (i) nucleotide position +356 in intron 4 (that is,with initial base of intron 4 as position +1) of EDN1 (rs2071942) or(ii) nucleotide position +61 in exon 5 (that is, with initial base ofexon 5 as position +1) of EDN1 (rs5370) encoding a polymorphism at aminoacid 198 of EDN1 protein, wherein the patient is being considered fortreatment with a β-adrenergic receptor targeting agent.

An “endothelin gene system member” of the present invention may be anygene that encodes an endothelin protein or encodes a protein thatdirectly operates on, or interacts with, or alters expression orregulation of, an endothelin. Non-limiting examples of endothelin genesystem members include any endothelin gene, any endothelin receptor(EDNR) gene, or any endothelin converting enzyme (ECE) gene. Inparticular embodiments, the endothelin gene system member is anendothelin gene. In certain embodiments, the endothelin gene is EDN1,EDN2, or EDN3. In particular embodiments, the endothelin gene is EDN1.In certain embodiments, the endothelin gene system member is anendothelin receptor gene, such as EDNRA or EDNRB. In certainembodiments, the endothelin gene system member is an endothelinconverting enzyme gene, such as ECE1 or ECE2. Proteins encoded byendothelin gene system members are also encompassed by the presentinvention.

As used herein, a “β-adrenergic receptor targeting agent” refers to asubstance and/or pharmacological agent that interacts with one or moremembers of the β-adrenergic receptor protein system. In certainembodiments, the β-adrenergic receptor targeting agent is a β-blocker.The β-blocker may be a non-selective β-blocker, as described below. Incertain embodiments, the non-selective β-blocker is selected from thegroup consisting of bucindolol, carteolol, carvedilol, carvedilolphosphate, nadolol, prebutolol sulfate, pindolol, propanololhydrochloride, sotalol hydrochloride, or rimolol maleate or a selectiveβ-blocker, as described below. The β-blocker may be a selectiveβ-blocker, as described below. In certain embodiments, the selectiveβ-blocker is selected from the group consisting of acebutolol, atenolol,betaxolol hydrochloride, bisoprolol, esmolol, metaprolol succinate, ormetoprolol tartrate. In particular embodiments, the β-adrenergicreceptor targeting agent is bucindolol. In particular embodiments, theβ-adrenergic receptor targeting agent is specifically not bucindolol.

The present invention is also concerned with obtaining informationregarding a polymorphism in an endothelin gene system member directly oras deduced by determining the nucleotide sequence at a certain position,and prescribing or administering a β-adrenergic receptor targeting agentbased on the obtained information. Such certain positions may include,for example, (i) nucleotide position +356 in intron 4 of EDN1(rs2071942) and/or (ii) nucleotide position +61 in exon 5 of EDN1(rs5370) encoding a polymorphism at amino acid 198 of EDN1 protein. Itwill be understood that cognate nucleic acids for proteins encoded byendothelin gene system members include the mRNA transcript encoding theprotein, both strands of any cDNA generated from the mRNA transcript,and both strands of the genomic DNA for the endothelin gene systemmember genes.

The invention provides a method for determining whether a β-adrenergicreceptor targeting agent should be prescribed to a patient wherein theidentity of a polymorphic nucleotide or amino acid site of an endothelingene system member is determined and based on the results of thatdetermination, a β-adrenergic receptor targeting agent is eitherprescribed or not. Similarly, based on the genotype, another medicationmay be prescribed for patient with the unfavorable genotype, so as toattempt to gain improved clinical response. In both scenarios, drugtreatment decisions are based on the endothelin gene system membergenotype of the patient.

Any method of the present invention may be employed with respect to apatient who has symptoms of or is suffering from a medical condition. Asused herein, “medical condition” includes but is not limited to anycondition or disease manifested as one or more physical and/orpsychological symptoms for which treatment is desirable, and includespreviously and newly identified diseases and other disorders havingsimilar pathophysiological states. For example, a medical condition maybe a cardiovascular disease. As used herein, a “cardiovascular disease”is any abnormal condition characterized by the dysfunction of the heartor blood vessels. Some examples of cardiovascular diseases aredisclosed, e.g., in Yale University School of Medicine Heart Book,Chapter 23, Cardiovascular Drugs, Apr. 16, 1999; Mosby's Medical,Nursing, & Allied Health Dictionary, 1998; and Stedman's MedicalDictionary, 1990. Non-limiting examples of medical conditions include,heart failure (HF), cardiac arrhythmias, hypertension, dilatedcardiomyopathy and ischemic heart disease (cardiomyopathy, angina,myocardial infarction). In certain embodiments, the patient has symptomsof or has been diagnosed with a medical condition comprising heartfailure or cardiomyopathy. In certain embodiments, the patient hassymptoms of or has been diagnosed with heart failure. In certainembodiments, the patient has symptoms of or has been diagnosed withdilated cardiomyopathy. The present invention further identifiespatients that will positively respond to treatment using a β-adrenergicreceptor targeting agent.

In some embodiments, methods include identifying a patient possibly inneed of treatment with a β-adrenergic receptor targeting agent, such asa β-blocker. A patient for which a β-adrenergic receptor targeting agentis being considered as a treatment option may have symptoms of or mayhave been diagnosed with a medical condition, as described herein. Incertain embodiments, the patient has symptoms of or has been diagnosedwith cardiomyopathy, such as dilated cardiomyopathy. In particularembodiments, a patient has symptoms of or has been diagnosed with heartfailure. The heart failure may be considered advanced heart failure,though the invention may not be limited to such patients. The term“advanced heart failure” is used according to its ordinary and plainmeaning in the field of cardiology. In some embodiments, a patient beingprescribed a β-adrenergic receptor targeting agent may have class III orclass IV heart failure according to the NYHA classification system. TheNYHA classification system is one evaluation system; however, it iscontemplated that the invention is not limited in this way and that thisis meant to be illustrative rather than limiting. Patients may beclassified by another such system. It is further contemplated thatpatients may be classified by a different methodology but that theinvention would be implemented similarly.

In other embodiments, however, a patient may have signs or symptoms ofheart failure but not advanced heart failure. In such a situation thepatient may have been or may be characterized as a class I or II heartfailure patient according to the NYHA classification system. In theseembodiments, the patient may be genotyped for a polymorphism asdescribed herein, in which case a person is a candidate for β-adrenergicreceptor targeting agent treatment. Consequently, methods of theinvention can involve preventing heart failure in a patient bydetermining whether the patient has a polymorphism as described hereinand administering a β-adrenergic receptor targeting agent if s/he does.Certain patients might be particularly suited for this including, butnot limited to, those patients with symptoms of heart failure, with riskfactors of heart failure, or with a familial or prior history of heartfailure.

A further method of the present invention contemplates a method forevaluating whether a heart failure patient will respond positively to aβ-adrenergic receptor targeting agent comprising determining (i) thepresence or absence of a polymorphism at nucleotide position +356 inintron 4 of EDN1 (rs2071942) of the patient, and/or (ii) the presence orabsence of a polymorphism at nucleotide position +61 in exon 5 of EDN1(rs5370).

In methods of the present invention, determining the presence or absenceof at least one polymorphism may be performed via any method known tothose of skill in the art. Those of skill in the art readily understandthat the coding sequence of a gene refers to the strand of the gene thatis used for transcription of messenger RNA. The sequence of the codingsequence is complementary to the sequence of the transcribed transcript.Because of the complementary nature of sequences between a codingsequence and a noncoding sequence, the sequence of any coding sequencecan be determined by knowing the sequence of the transcript, thenoncoding strand, or the encoded protein. The nucleic acid sequence atthat position in one or both alleles can be determined by a number ofways known to those of skill in the art. In certain embodiments,determining the presence or absence of at least one polymorphismcomprises pyrosequencing, chain terminating sequencing, restrictiondigestion, allele-specific polymerase reaction, single-strandedconformational polymorphism analysis, genetic bit analysis, temperaturegradient gel electrophoresis, ligase chain reaction, or microarrayhybridization.

Alternatively, the sequence of a protein encoded by an endothelin genesystem member may be evaluated. This evaluation may take place via anymethod known to those of skill in the art. Methods for determining thesequence at a particular position in a protein are well known and mayinvolve using, for example, an antibody, high pressure liquidchromatography, or mass spectroscopy. In certain embodiments, the aminoacid at position 198 in one or more of the patient's EDN1 proteins isknown. It is contemplated that any sample evaluated from the patientwill contain multiple endothelin gene system member proteins that can beanalyzed.

In any method of the present invention, a patient's genotype atnucleotide position +356 in intron 4 of EDN1 (rs2071942) may be known.Alternatively, in any method of the present invention, the patient'sgenotype at nucleotide position +356 in intron 4 of EDN1 (rs2071942) isunknown. In certain embodiments, the patient may be determined to havean adenosine or a guanine at nucleotide position +356 in intron 4 ofEDN1 (rs2071942).

In any method of the present invention, the patient's genotype atnucleotide position +61 in exon 5 of EDN1 (rs5370) may be known.Alternatively, in any method of the present invention, the patient'sgenotype at nucleotide position +61 in exon 5 of EDN1 (rs5370) isunknown. In any method of the present invention, the nucleotide atposition +61 in exon 5 of EDN1 (rs5370) is part of a codon that encodesa lysine or an asparagine. In certain embodiments, the patient'sgenotype at nucleotide position +356 in intron 4 of EDN1 (rs2071942) andthe patient's genotype at nucleotide position +61 in exon 5 of EDN1(rs5370) are both unknown.

It is contemplated that in the context of the present invention,typically a medical practitioner (e.g., doctor, nurse, or their staff)will be evaluating whether to prescribe or administer a patient aβ-adrenergic receptor targeting agent and in making that evaluation, thepractitioner will order one or more tests regarding one or more of thepatient's endothelin gene system member alleles or their encodedproteins. Accordingly, any method of the present invention may furthercomprise obtaining a patient history. For example, in methods wherein atleast one polymorphism is at (i) nucleotide position +356 in intron 4 ofEDN1 (rs2071942) or (ii) nucleotide position +61 in exon 5 of EDN1(rs5370) encoding a polymorphism at amino acid 198 of EDN1 protein,wherein the individual is being considered for treatment with aβ-adrenergic receptor targeting agent, a patient history regarding (i)or (ii) may be obtained. Any method of the present invention may furthercomprise preparing a report containing the results of determining (i) or(ii). Such a report may identify the patient by name, social securitynumber, and/or other identification number or qualifier. It may alsocontain the actual data as a result of the determination or a summary ofthat data.

Other information may also be considered in determining whether aβ-adrenergic receptor targeting agent is an appropriate drug for thepatient. This may include race, gender, age, previous surgeries, heartfailure stage, patient history regarding cardiovascular disease,diagnosis of other diseases or conditions, risks for other diseases orcondition, drug allergies, drug toxicity, and/or other medications beingtaken.

In any method of the present invention, a biological sample may beobtained from a patient. Any biological sample may be obtained, such asone that contains DNA. A biological sample is a sample that containsbiological material such as all or part of an organ, tissue, cells,nucleic acids, proteins, or other such macromolecules and substances.The sample may include sputum, serum, blood, plasma, spinal fluid,semen, lymphatic fluid, urine, stool, pleural effusion, ascites, atissue sample, tissue biopsy, cell swab, or a combination thereof. Inother embodiments of the invention, a sample may include cells that arefrom lung, skin, muscle, liver, renal, colon, prostate, breast, brain,bladder, small intestine, large intestine, cervix, stomach, pancreas,testes, ovaries, bone, marrow, or spine. In some embodiments, the sampleis a whole blood, plasma or serum sample, while in other embodiments,the sample is obtained by lavage, smear, or swab of an area on or in thepatient. In certain embodiments, the biological sample is a bloodsample. In particular embodiments, a biological sample is blood, saliva,or skin.

To achieve certain methods of the present invention, a medicalpractitioner may obtain the biological sample for evaluation. The samplemay be analyzed by the practitioner, or it may be sent to an outside orindependent laboratory. The medical practitioner may or may not becognizant of what information regarding the patient's endothelin genesystem member(s) is being obtained. In any of these circumstances, themedical practitioner may consequently know the relevant information thatwill allow him or her to determine whether β-adrenergic receptortargeting agent therapy is an appropriate medicinal option. It iscontemplated that, for example, a laboratory conducts the test todetermine that patient's genotype such that its personnel also know theappropriate information. They may report back to the practitioner withthe specific result of the test performed or the laboratory may simplyreport that a β-adrenergic receptor targeting agent is an appropriatedrug based on the laboratory results.

It is contemplated that, in certain situations, a patient may begenotyped for one of these polymorphisms and then a subsequentdetermination is made with respect to the other polymorphism; in thisscenario, two different samples may be evaluated. Alternatively, asingle sample may be obtained and evaluated for two separatepolymorphisms. Furthermore, it is contemplated that the invention alsoconcerns performing a diplotype analysis or obtaining the results of adiplotype analysis.

In some embodiments of the invention, the sequence of a patient'sendothelin gene system member and/or a protein encoded thereby mayalready have been evaluated. It is contemplated that this analysis mayhave been done prior to the patient being considered for treatment witha β-adrenergic receptor targeting agent or as part of a generalexamination. For example, the sequence of a patient's endothelin genesystem member or a protein encoded thereby may be determined and enteredinto a database or entered into the patient's medical history. In thiscase, a medical practitioner may come to know what the sequence is byobtaining a patient history regarding the sequence at (i) nucleotideposition +356 in intron 4 of EDN1 (rs2071942) or (ii) nucleotideposition +61 in exon 5 of EDN1 (rs5370) encoding a polymorphism at aminoacid 198 of EDN1 protein.

In particular embodiments, any method as described herein furthercomprises administering a β-adrenergic receptor targeting agent, such asbucindolol, to the patient after determining (i) the nucleotide position+356 in intron 4 of EDN1 (rs2071942) or (ii) the nucleotide position +61in exon 5 of EDN1 (rs5370) encoding a polymorphism at amino acid 198 ofEDN1 protein, wherein the patient has an adenosine or a guanine atnucleotide position +356 in intron 4 of EDN1 (rs2071942), and/or thenucleotide at position +61 in exon 5 of EDN1 (rs5370) encodes a lysineor an asparagine at amino acid 198 of EDN1 protein.

In particular embodiments, any method as described herein may furthercomprise administering to a patient a β-adrenergic receptor targetingagent that is not bucindolol after determining (i) the nucleotideposition +356 in intron 4 of EDN1 (rs2071942) or (ii) the nucleotideposition +61 in exon 5 of EDN1 (rs5370) encoding a polymorphism at aminoacid 198 of EDN1 protein, wherein the patient has neither an adenosineor a guanine at nucleotide position +356 in intron 4 of EDN1 (rs2071942)nor a nucleotide at position +61 in exon 5 of EDN1 (rs5370) that encodesa lysine or an asparagine at amino acid 198 of EDN1 protein.

Other general aspects of the present invention contemplate a method fortreating a patient with a heart condition comprising administering tothe patient an effective amount of a β-adrenergic receptor targetingagent, wherein the patient has detectable EDN1 protein with anasparagine or lysine at amino acid 198 of EDN1 protein. The term“effective,” as that term is used in the specification and/or claims(e.g., “an effective amount”) means adequate to accomplish a desired,expected, or intended result. The β-adrenergic receptor targeting agentmay be of any type described herein, such as a selective or anon-selective β-blocker. In particular embodiments, the β-adrenergicreceptor targeting agent is bucindolol.

It is contemplated that not all of the patient's proteins will beevaluated in any embodiment of the invention but that a sample will beobtained and some of the proteins in the sample will be evaluated fortheir protein sequence. The same holds true for any evaluation of apatient's nucleic acids as well.

Another general aspect of the present invention contemplates a methodfor treating a patient with a heart condition comprising administeringto the patient an effective amount of a β-adrenergic receptor targetingagent, wherein the patient does not have detectable EDN1 protein with anasparagine or lysine at amino acid 198 of EDN1 protein. The β-adrenergicreceptor targeting agent may be of any type described herein, such as aselective or a non-selective β-blocker. In particular embodiments, theβ-adrenergic receptor targeting agent is bucindolol.

Yet another general aspect of the present contemplates a method fortreating a patient with a heart condition comprising administering tothe patient an effective amount of a β-adrenergic receptor targetingagent, wherein the patient homozygous for an adenosine at nucleotideposition +356 in intron 4 of EDN1 (rs2071942), or asparagine at aminoacid 198 of EDN1 protein. Methods of the invention thus involveprescribing or administering a β-adrenergic receptor targeting agent topatients who are homozygous in this regard, regardless of how it isdetermined that the patient has that genotype. The β-adrenergic receptortargeting agent may be of any type described herein, such as a selectiveor a non-selective β-blocker. In particular embodiments, theβ-adrenergic receptor targeting agent is bucindolol.

Other methods of the present invention contemplate a method for treatinga patient with a β-adrenergic receptor targeting agent comprising: (a)determining (i) the presence or absence of a polymorphism at nucleotideposition +356 in intron 4 of EDN1 (rs2071942) of the patient, and/or(ii) the presence or absence of a polymorphism at nucleotide position+61 in exon 5 of EDN1 (rs5370); and (b) either (i) prescribing aβ-adrenergic receptor targeting agent for the patient, wherein thepatient's genotype is homozygous for an adenosine at nucleotide position+356 in intron 4 of EDN1, or asparagine at amino acid 198 of EDN1protein; or (ii) not prescribing a β-adrenergic receptor targeting agentfor the patient, wherein the patient's genotype is not homozygous for anadenosine at nucleotide position +356 in intron 4 of EDN1 (rs2071942),or not an asparagine at amino acid 198 of EDN1 protein. The β-adrenergicreceptor targeting agent may be of any type described herein, such as aselective or a non-selective β-blocker. In particular embodiments, theβ-adrenergic receptor targeting agent is bucindolol.

In certain embodiments, a patient may be a non-Hispanic White or anon-Hispanic Black. Certain methods of the present invention comprisedetermining that a patient has an adenosine or a guanine at nucleotideposition +356 in intron 4 of EDN1 (rs2071942), and the nucleotide atposition +61 in exon 5 of EDN1 (rs5370) encodes a lysine or anasparagine at amino acid 198 of EDN1 protein. In certain embodiments ofsuch methods, the patient is a non-Hispanic White.

The present invention also provides devices and compositions for thedelivery of a β-adrenergic receptor targeting agent to an individual inneed of such therapy. Additionally, methods may involve administering orprescribing other therapeutic agents or performing a surgical or otherinterventional strategy for treating the patient.

The embodiments discussed with respect to methods may be implemented inuse of a β-adrenergic receptor targeting agent in the manufacture of amedicament.

The term “treatment” will be understood to refer to therapy with respectto a patient diagnosed with a medical condition, as described herein, orwith symptoms of a medical condition, as opposed to preventativemeasures. However, as discussed above, preventative measures are alsocontemplated by the present invention.

Any embodiment discussed with respect to one aspect of the inventionapplies to other aspects of the invention as well. This includesembodiments discussed with respect to the endothelin gene system membersdescribed herein. For example, any embodiment discussed with respect toEDN1, EDN2, EDN3, EDNRA, EDNRB and ECE1 genes, alleles, or proteins maybe implemented with respect to EDN1, EDN2, EDN3, EDNRA, EDNRB and ECE1genes, alleles, or proteins, and vice versa.

The embodiments in the Examples section are understood to be embodimentsof the invention that are applicable to all aspects of the invention.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

The words “comprise,” “comprising,” “include,” “including,” and“includes” when used in this specification and in the following claimsare intended to specify the presence of stated features, integers,components, or steps, but they do not preclude the presence or additionof one or more other features, integers, components, steps, or groupsthereof.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Time to the combined event of first heart failurehospitalization or death for endothelin-1 polymorphisms Lys198Asn (top)and IVS-4 G/A by genotype. Common homozygotes, heterozygotes and rarehomozygotes are depicted by the solid, dashed and dotted lines,respectively. The data are separated by treatment group with placebotreated and bucindolol treated subjects on the left and right,respectively. P-values are for the 2 df log-rank test. Event/number (forcommon homozygotes, heterozygotes and rare homozygotes, respectively)for Lys198Asn: (placebo: 36/89, 21/60, 3/9; bucindolol: 22/87, 20/55,7/8) and IVS-G/A: (placebo: 35/87, 23/63, 3/9; Bucindolol: 21/84, 21/58,7/8).

FIG. 2. Hazard ratios (HR) and 95% confidence intervals by treatment forthe effect of a one-allele change in genotype, unadjusted for multiplecomparisons for each endothelin system gene. EDN1-endothelin-1;EDNRA-endothelin receptor A; EDNRB-endothelin receptor B;ECE1-endothelin converting enzyme-1. Dashed and solid lines indicate theHRs for placebo and bucindolol groups, respectively. Values greater thanone indicate greater risk associated with the less common allele.

FIG. 3. Location of EDN1 polymorphisms studied. The genomic structure ofthe five exons (boxes) of the endothelin-1 gene is depicted (top) alongwith the four EDN1 polymorphisms analyzed. The Lys198Asn and IVS-4 G/Apolymorphisms associated with bucindolol response are underlined. TheLys198Asn polymorphism is translated into the preproendothelin peptide,but is removed during processing into big endothelin.

FIG. 4. Partial sequence of genomic DNA for EDN1 based on EnsemblENSG00000078401. The first SNP is rs2071942 in intervening sequence 4(IVS-4), noted as [g/a]. The second SNP is rs5370 in exon 5, noted as[G/C]. Exons are in upper case; introns are in lower case. Exon 4 isunderlined. Exon 5 is are all in capital letters (both underlined andnot underlined). Primers are highlighted in gray.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As noted above, β-blocker therapy is standard treatment for a variety ofmedical conditions, such as cardiovascular conditions. Observedvariation in response to medication is frequently mediated by geneticfactors (Wilkinson, 2005; Evans and Relling, 2004; Weinshilboum, 2003).In the case of heart failure, the limited number of DNA banksestablished with large prospective trials has historically limiteddiscovery of pharmacogenetic markers of drug response. More recentlyhowever, success in identifying pharmacogenetic predictors of drugresponse in heart failure have been reported (Liggett et al., 2006).

The endothelin gene system (EGS) is a candidate modifier pathway forheart failure (Nambi et al., 2001; Levin, 1995; Miyauchi and Masaki,1999). Short endothelin peptides bind to endothelin receptors and arepotent mediators of vasoconstriction, endothelial cell growth andvascular tone (Dzau, 1993; Yanagisawa et al., 1988). Changes in cardiacinotrophy and chronotrophy are mediated also through binding ofendothelin to endothelin receptors A and B. Endothelin-1 is thepredominant isoform expressed in the human heart and promotescardiomyocyte contractility and hypertrophy (Yanagisawa et al., 1988;Inoue et al., 1989). Genetic variation in endothelin has been associatedwith blood pressure, left-ventricular hypertrophy and heart failure(Stevens and Brown, 1995; Vasku et al., 2002; Brugada et al., 1997).Moreover, biologically active endothelins interact with other‘neurohormonal’ systems, including adrenergic and antiogensin systems,by pharmacologic crosstalk (Krum et al., 1996; Spieker et al., 2001;Spieker and Luscher, 2003; Giannessi et al., 2001).

Prior genetic studies of EGS single nucleotide polymorphisms (SNPs)focused on hypertension, arterial stiffness, vascular reactivity,arrhythmias, myocardial infarction, and heart failure (Iemitsu et al.,2006; Kozak et al., 2004; Jin et al., 2003; Iglarz et al., 2002; Tiretet al., 1999; Herrmann et al., 2001; Charron et al. 1999; Nicaud et al.,1999). The majority of these studies were not prospective and none ofthe known EGS polymorphisms have been tested for pharmacogenetic effectsin heart failure. The present inventors hypothesized that response toβ-blocker medication might be mediated by EGS variation. EGS SNPs weretherefore studied for evidence of pharmacogenetic associations withclinical outcomes from a large, prospective, β-blocker heart failuretrial reported previously (Beta-Blocker Evaluation of Survival TrialInvestigators, 2001).

As shown in the Examples below, alleles at two EDN1 SNPs, Lys198Asn andG/A (IVS-4), commonly appearing in one of two complementary haplotypes,showed a strong pharmacogenetic interaction with β-adrenergic receptortargeting agent therapy on time to first heart failure hospitalizationor all cause mortality. One result observed in these experiments is thenovel identification of a haplotype associated with clinical response toa β-adrenergic receptor targeting agent in dilatedcardiomyopathy—specifically, a β-blocker. The observed effect wasconfined to the treated group, with no significant effect in the placebogroup, consistent with a true pharmacogenetic effect where drug‘exposure’ elicits different outcomes based on the genetic background. A‘dose-response’ trend by haplotype was observed, with subjectshomozygous for the rare haplotype having the highest hazard ratios (HRs)as compared to the relative ‘protective’ effect of the common haplotype.A relative benefit of β-blocker therapy for the 56% of subjectshomozygous for the common haplotype identifies a large subpopulationwhere β-blocker therapy appears most beneficial. Contrasted against thisgroup are the 5.5% of subjects, homozygous for the rare complementaryhaplotype, for whom the combined endpoint hazard rate was almost threetimes greater on bucindolol than on placebo.

Bucindolol is a β-blocking agent with sympatholytic and vasodilatorproperties. Although it is not yet approved for clinical heart failuretherapy, variation in outcomes in sub-populations of the study suggestedto the inventors that underlying genetic differences may be responsiblefor observed differences in efficacy.

EDN1 G/A (IVS-4) and Lys198Asn are in tight linkage disequilibrium,which agrees with other published data and makes it impossible todistinguish which SNP is responsible for the observed effect. Stronglinkage disequilibrium is also present between these SNPs and thers1800543 variant in intron 3, which was not tested (Diefenbach et al.,2006). Genotyping of G/A IVS-4, Lys198Asn and rs1800543 largely definethe same haplotype block extending from the beginning of intron 2 toexon 5. Three additional intronic SNPs not tested (rs:2070669, 1800543,641347) and the Glu106 variant, which was not significant after multipletesting, also are contained in this haplotype block (Diefenbach et al.,2006).

The G/A (IVS-4) variant is at position +356 in intron 4 and does notlocalize to a known functional splicing or regulatory site (FIG. 3). Itis located 6 base pairs downstream from an in silico predictedregulatory region using the ESPERR algorithm (Kolbe et al., 2004; Kinget al., 2005). The SNP does not alter splicing in an in silico model(GeneSplicer, world wide web at .tigr.org/), thus an obvious functionaleffect is lacking. Previous studies of this SNP showed a modestinfluence on variability of left ventricular hypertrophy 32 and doubleheterozygosity of IVS-4 G/A and rs1800997 predicted lower big endothelinlevels in one study of chronic heart failure (Vasku et al., 2002). Thehighly linked rs1800543 also shows no predicted effect on splicing.

The nonsynonymous change at position 198 where a basic, charged lysineis replaced with a smaller, polar asparagine residue may affectsecondary protein structure by extending regional coiled structure (GORIV and PSIPRED algorithms: world wide web at npsa-pbil.ibcp.fr/ andbioinf.cs.ucl.ac.uk/, respectively). Serum endothelin-1 levels and bigendothelin levels predict clinical heart failure phenotypes, andelevations correlate directly with heart failure severity and poorerprognosis and inversely with ejection fraction and cardiac index (Wei etal., 1994; McMurray et al. 1992; Hiroe et al., 1991). The differences inendothelin levels are mediated by levels of its precursor,big-endothelin (Wei et al., 1994). Without being bound by theory, theinventors suppose it is possible that the Asn198 variant subtly altersbig-endothelin levels, stability, or conversion to endothelin-1 byendothelin converting enzyme (ECE). Compatible with this model are datafrom pregnant women and a separate hypertensive population where Asn198alleles were predictive of higher serum endothelin levels (Tanaka etal., 2004; Barden et al., 2001).

Accordingly, the present invention, in part, concerns methods thatutilize the genetic relationship between the polymorphisms at (i)nucleotide position +356 in intron 4 of EDN1 (rs2071942) or (ii)nucleotide position +61 in exon 5 of EDN1 (rs5370) encoding apolymorphism at amino acid 198 of EDN1 protein and β-adrenergic receptortargeting agent therapy.

I. ENDOTHELIN GENE SYSTEM MEMBERS

As mentioned above, endothelins bind to endothelin receptors and arepotent mediators of vasoconstriction, endothelial cell growth andvascular tone. In a healthy individual, a delicate balance betweenvasoconstriction and vasodilation is maintained by endothelin,calcitonin and other vasoconstrictors on the one hand and nitric oxide,prostacyclin and other vasodilators on the other. Elevated activation ofthe endothelin signaling pathway also induces cell proliferation and/orsurvival and is implicated in a variety of malignancies (Zhang et al.,2006). A variety of endothelin gene system members and the proteins theyencode are commercially available and/or have been prepared in theliterature (e.g., Bachem AG, Switzerland; Invitrogen Corp., Carlsbad,Calif.; U.S. Pat. No. 6,066,502).

There are three isoforms of endothelin, each containing 21 amino acids:EDN1, EDN2 and EDN3. Two of the 21 amino acids differ between EDN1 andEDN2, and six between EDN1 and EDN3 (Inoue et al. 1989). Each endothelinis encoded by a separate preproendothelin, and each is subsequentlyproduced via its own intermediate, referred to as big endothelin. Theendothelins show varying regions of expression. Human EDN1 mRNA is foundin several organs, including the brain, kidney, lung, uterus andplacenta; human EDN2 mRNA is found abundantly in renal medulla and thejejunum, and plays a role in the ovulatory process (Ko et al., 2006);and human EDN3 mRNA is found abundantly in the jejunum and adrenalgland, as well as in the brain, spleen and renal medulla (Arinami etal., 1991).

It is necessary for a cell to express endothelin-converting enzyme (ECE)to catalyze the conversion of inactive forms of endothelins (163 aminoacids) into bioactive endothelins (21 amino acids) to produce bioactiveendothelins. There are at least two identified ECE genes, ECE1 and ECE2,and several isoforms of each have been detected. (Valdenaire et al.,1999; Meidan et al., 2005; Ikeda et al., 2002). ECE2 was discovered in1995 as a novel member of the ECE1 gene family; the two gene productsshare 59% amino acid identity (Emoto and Yanagisawa, 1995; Turner etal., 2001).

Two key receptor types are known, EDNRA and EDNRB, which areG-protein-coupled receptors. EDNRA receptors are found in the smoothmuscle tissue of blood vessels, and binding of endothelin to EDNRAincreases vasoconstriction and the retention of sodium. These actionslead to increased blood pressure. EDNRB is primarily located on theendothelial cells that line the interior of the blood vessels. Whenendothelin binds to EDNRB receptors, this leads to increased natriuresisand diuresis and the release of nitric oxide (also endothelium-derivedrelaxing factor), all mechanisms that lower the blood pressure.

II. β-ADRENERGIC RECEPTOR TARGETING AGENTS

A. β-Adrenergic Receptors

The adrenergic receptors (AR) are a class of G protein-coupled receptorsthat are targets of the catecholamines. Adrenergic receptorsspecifically bind and are activated by their endogenous ligands, thecatecholamines adrenaline and noradrenaline (also called epinephrine andnorepinephrine, respectively). There are at least nine sub-types ofadrenergic receptors (Dohlman et al., 1991; and Liggett et al., 1993),and they are typically categorized as α-adrenergic (e.g., α₁, α₂) orβ-adrenergic (e.g., β₁, β₂ and β₃).

The β₁ adrenergic receptor (β₁-AR) is the principle subtype expressed oncardiac myocytes. The human heart expresses both the β₁-AR and the β₂-ARsubtypes (Bristow et al, 1986; Bristow et al., 1988). Each receptormediates positive inotropic and chronotropic responses to endogenouscatecholamines and exogenously administered agonists (Bristow et al.,1986; Brodde et al., 1986; Brodde et al., 1992). β₁-AR triggers theheart's contractile response when activated, as it is by norepinephrine.In addition, the β₁ receptor has a central role in the progression ofcardiomyopathy and other disease pathways. Increased activation of thisreceptor and its associated myopathic and arrhythmic pathways plays amajor role in the natural history of heart failure. Once thecardiomyopathic process has begun, chronic β₁-adrenergic activationaccelerates disease progression, as the failing heart tries tocompensate for its impaired functioning by releasing more norepinephrineand increasing β₁-receptor signaling. The theory of β-receptor blockaderests in part on counteracting this cardiomyopathic pathway by blockingthe β₁-receptor and reducing norepinephrine signaling.

β₁-AR has been cloned and sequenced (Frielle et al., 1987). The gene hasbeen localized to chromosome q24-q26 of chromosome 10 (Yang-Feng et al.,1990). The human β₁AR has a deduced amino acid sequence of 477 aminoacids.

The β₂ adrenergic receptor (β₂-AR) is found in the lung, smooth muscle,cerebellum and skeletal muscle. The receptor is involved in respiratorydiseases such as chronic bronchitis, emphysema, acute respiratorydistress syndrome and asthma. For example, in the lung, β₂-AR agonistscause bronchiole dilation and thus can be useful in the treatment ofasthma. β₂-AR also promotes the release of insulin and is furtherresponsible for relaxation of uterine muscle. Certain treatments ofpremature labor target β₂-AR. Other conditions associated with β₂-ARinclude hypertension, congestive heart failure and cardiovascular shock.β₂-AR agonists may also be useful for treatment of neurologicaldisorders.

The gene encoding the human β₂-AR has also been cloned and sequenced(Kobilka et al., 1987). It is an intronless gene that has been localizedto q31-q32 of chromosome 5. The deduced amino acid sequence consists of413 amino acids, with seven clusters of hydrophobic residues thought torepresent transmembrane spanning domains. The N-terminus isextracellular, containing two sites for asparagine-linked glycosylation.The transmembrane spanning domains are connected by three extracellularand three intracellular loops. The C-terminus is intracellular.

β₃-AR is less well characterized as β₁-AR and β₂-AR. This receptorcontains 402 amino acids and is capable of activating adenylate cyclasein the presence of an agonist. Through a pharmacological comparison ofthe activation of adenylate cyclase in the presence of agonists and thereaction towards different antagonists, this receptor was shown todiffer from β₁-AR and β₂-AR. The expression of β₃-AR has been reportedin various tissues such as adipose tissue and tissues of the digestivetract (esophagus, colon and gallbladder) (Bond et al., 1988); Coleman etal., 1987); Bianchetti et al., 1990); Granneman et al., 1991). β₃-ARagonists have been pursued as antiobesity and antidiabetic agents, andmay also find in controlling the frequent urge of urination (see U.S.Patent Publication No. 2003/0176412). The cloning and initial sequencingof the human and mouse β₃-AR genes have been previously described(Nahmias et al., 1991; Emorine et al., 1989).

B. β-Adrenergic Receptor Targeting Agents

β-blockers are a class of drugs used for various indications, butparticularly for the management of cardiac arrhythmias andcardioprotection after myocardial infarction. Although β-blockers wereonce contraindicated in congestive heart failure, as they have thepotential to worsen the condition, studies in the late 1990s showedtheir positive effects on morbidity and mortality in congestive heartfailure (Hjalmarson et al., 2000; Leizorovicz et al., 2002; Packer etal., 2002). Although no β-blocker is approved for anxiolytic use by theU.S. Food and Drug Administration, some people use β-blockers to avoidstage fright and tremor during public performance and auditions. Indeed,some Olympic marksmen take β-blockers to provide more aiming timebetween heart beats since these drugs lower one's heart rate. Presently,more than ten different β-blockers are available, all by prescription.

Acting as antagonists of β-adrenergic receptors, β-blockers block theaction of endogenous catecholamines (epinephrine (adrenaline) andnorepinephrine (noradrenaline), in particular) on β-adrenergicreceptors, part of the sympathetic nervous system that mediates the“fight or flight” response. This action slows the nerve impulses thattravel through the heart. As a result, the heart beats more slowly andless strongly, and blood pressure falls. β-blockers also block impulsesthat can cause arrhythmia, and stop blood vessels around the brain fromwidening so easily, helping to prevent migraines.

Non-limiting examples of a β-blockers include acebutolol (sectral),alprenolol, amosulalol, arotinolol, atenolol, befunolol, betaxolol,bevantolol, bisoprolol, bopindolol, bucumolol, bufetolol, bufuralol,bunitrolol, bupranolol, butidrine hydrochloride, butofilolol, carazolol,carteolol, carvedilol, celiprolol, cetamolol, cloranolol, dilevalol,epanolol, esmolol (brevibloc), indenolol, labetalol, levobunolol,mepindolol, metipranolol, metoprolol, moprolol, nadolol, nadoxolol,nifenalol, nipradilol, oxprenolol, penbutolol, pindolol, practolol,pronethalol, propanolol (inderal), sotalol (betapace), sulfinalol,talinolol, tertatolol, timolol, toliprolol and xibinolol. In certainaspects, the β-blocker comprises an aryloxypropanolamine derivative.Non-limiting examples of aryloxypropanolamine derivatives includeacebutolol, alprenolol, arotinolol, atenolol, betaxolol, bevantolol,bisoprolol, bopindolol, bunitrolol, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, epanolol, indenolol, mepindolol,metipranolol, metoprolol, moprolol, nadolol, nipradilol, oxprenolol,penbutolol, pindolol, propanolol, talinolol, tertatolol, timolol andtoliprolol.

In certain aspects, “derivative” refers to a chemically modifiedcompound that still retains the desired effects of the compound prior tothe chemical modification. Such derivatives may have the addition,removal, or substitution of one or more chemical moieties on the parentmolecule. Non-limiting examples of the types modifications that can bemade to the compounds and structures disclosed herein include theaddition or removal of lower alkyls such as methyl, ethyl, propyl, orsubstituted lower alkyls such as hydroxymethyl or aminomethyl groups;carboxyl groups and carbonyl groups; hydroxyls; nitro, amino, amide, andazo groups; sulfate, sulfonate, sulfono, sulfhydryl, sulfonyl,sulfoxido, phosphate, phosphono, phosphoryl groups, and halidesubstituents. Additional modifications can include an addition or adeletion of one or more atoms of the atomic framework, for example,substitution of an ethyl by a propyl; substitution of a phenyl by alarger or smaller aromatic group. Alternatively, in a cyclic or bicyclicstructure, heteroatoms such as N, S, or O can be substituted into thestructure instead of a carbon atom to generate, for example, aheterocycloalkyl structure. The present invention specificallycontemplates employing β-adrenergic receptor targeting agent derivativesin the methods described herein.

As agents that target β-adrenergic receptors, the present inventioncontemplates all categories of β-blockers, including selective andnon-selective β-blockers. Such agents are typically commerciallyavailable. The present invention also specifically contemplatesβ-blockers that have yet to be developed.

β-blockers that affect both β₁- and β₂-adrenergic receptors are termed“non-selective β-blockers.” Non-limiting examples of non-selectiveβ-blockers that may be employed in the present invention includealprenolol, bucindolol, carteolol, carvedilol, labetalol, levobunol,mepindolol, metipranolol, nadolol, oxprenolol, penbutolol, pindolol,prebutolol sulfate, propanolol, sotalol, rimolol and timolol.

“Selective β-blockers” primarily affect β₁ receptors. Selectiveβ-blockers gradually become less selective at higher doses. Non-limitingexamples of selective β-blockers that may be employed in the presentinvention include acebutolol, atenolol, betaxolol, hydrochloride,bisoprolol, celiprolol, esmolol, metaprolol, metoprolol, nebivolol andtimolol.

III. ANALYSIS OF POLYMORPHISMS

Certain genetic variants of the present invention are in coding regionsof endothelin gene system members and therefore typically affect theencoded proteins; as such, the presence of a endothelin gene systemmember polymorphism can be determined from either the sequence of thenucleic acid or the protein. However, not every polymorphism in a generesults in a difference in the encoded protein: for example, the G/A(IVS-4) polymorphism described herein does not the sequence of aprotein. The terms “endothelin gene system member polymorphism,”therefore, is a term of art and refers to polymorphisms in the nucleicacid or amino acid sequence of a endothelin gene system member gene orgene product. A variety of different methodologies can be employed forthe purpose of detecting polymorphisms in genes or gene products.

A. Nucleic Acids

Certain embodiments of the present invention concern various nucleicacids, including amplification primers, oligonucleotide probes, andother nucleic acid elements involved in the analysis of genomic DNA. Incertain aspects, a nucleic acid comprises a wild-type, a mutant, or apolymorphic nucleic acid.

For the purposes of identifying the location of a polymorphism, thefirst nucleotide of the start codon of the coding region (the adenine ofthe ATG in a DNA molecule and the adenine of the AUG in an RNA molecule)of an endothelin gene system member is considered nucleotide “1” and thenumbers progress according along the coding sequence. Similarly, thefirst amino acid of the translated protein product (the methionine) isconsidered amino acid “1.”

The term “nucleic acid” is well known in the art. A “nucleic acid” asused herein will generally refer to a molecule (i.e., a strand) of DNAor RNA comprising a nucleobase. A nucleobase includes, for example, anaturally occurring purine or pyrimidine base found in DNA (e.g., anadenine “A,” a guanine “G,” a thymine “T” or a cytosine “C”) or RNA(e.g., an A, a G, an uracil “U” or a C). The term “nucleic acid”encompass the terms “oligonucleotide” and “polynucleotide,” each as asubgenus of the term “nucleic acid.” The term “oligonucleotide” refersto a molecule of between about 3 and about 100 nucleobases in length.The term “polynucleotide” refers to at least one molecule of greaterthan about 100 nucleobases in length. A “gene” refers to coding sequenceof a gene product, as well as introns and the promoter of the geneproduct.

In some embodiments, nucleic acids of the invention comprise or arecomplementary to all or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570,580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710,720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850,860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990,1000, 1100, 1165, 1200, 1300, 1400, 1500 or more contiguous nucleotides,or any range derivable therein, of an endothelin gene system member cDNAsequence comprising a polymorphism as described herein. One of skill inthe art knows how to design and use primers and probes for hybridizationand amplification, including the limits of homology needed to implementprimers and probes.

These definitions generally refer to a single-stranded molecule, but inspecific embodiments will also encompass an additional strand that ispartially, substantially, or fully complementary to the single-strandedmolecule. Thus, a nucleic acid may encompass a double-stranded moleculeor a triple-stranded molecule that comprises one or more complementarystrand(s) or “complement(s)” of a particular sequence comprising amolecule. As used herein, a single stranded nucleic acid may be denotedby the prefix “ss”, a double stranded nucleic acid by the prefix “ds”,and a triple stranded nucleic acid by the prefix “ts.”

In particular aspects, a nucleic acid encodes a protein, polypeptide, orpeptide. In certain embodiments, the present invention concerns novelcompositions comprising at least one proteinaceous molecule. As usedherein, a “proteinaceous molecule,” “proteinaceous composition,”“proteinaceous compound,” “proteinaceous chain,” or “proteinaceousmaterial” generally refers, but is not limited to, a protein of greaterthan about 20 amino acids or the full length endogenous sequencetranslated from a gene; a polypeptide of greater than about 100 aminoacids; and/or a peptide of from about 3 to about 100 amino acids. Allthe “proteinaceous” terms described above may be used interchangeablyherein.

1. Preparation of Nucleic Acids

A nucleic acid may be made by any technique known to one of ordinaryskill in the art, such as for example, chemical synthesis, enzymaticproduction or biological production. Non-limiting examples of asynthetic nucleic acid (e.g., a synthetic oligonucleotide), include anucleic acid made by in vitro chemical synthesis using phosphotriester,phosphite or phosphoramidite chemistry and solid phase techniques suchas described in European Patent 266,032, incorporated herein byreference, or via deoxynucleoside H-phosphonate intermediates asdescribed by Froehler et al., 1986 and U.S. Pat. No. 5,705,629, eachincorporated herein by reference. In the methods of the presentinvention, one or more oligonucleotide may be used. Various differentmechanisms of oligonucleotide synthesis have been disclosed in forexample, U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566,4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which isincorporated herein by reference.

A non-limiting example of an enzymatically produced nucleic acid includeone produced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of anoligonucleotide described in U.S. Pat. No. 5,645,897, incorporatedherein by reference. A non-limiting example of a biologically producednucleic acid includes a recombinant nucleic acid produced (i.e.,replicated) in a living cell, such as a recombinant DNA vectorreplicated in bacteria (see for example, Sambrook et al. 2001,incorporated herein by reference).

2. Purification of Nucleic Acids

A nucleic acid may be purified on polyacrylamide gels, cesium chloridecentrifugation gradients, chromatography columns or by any other meansknown to one of ordinary skill in the art (see for example, Sambrook etal., 2001, incorporated herein by reference). In some aspects, a nucleicacid is a pharmacologically acceptable nucleic acid. Pharmacologicallyacceptable compositions are known to those of skill in the art, and aredescribed herein.

In certain aspects, the present invention concerns a nucleic acid thatis an isolated nucleic acid. As used herein, the term “isolated nucleicacid” refers to a nucleic acid molecule (e.g., an RNA or DNA molecule)that has been isolated free of, or is otherwise free of, the bulk of thetotal genomic and transcribed nucleic acids of one or more cells. Incertain embodiments, “isolated nucleic acid” refers to a nucleic acidthat has been isolated free of, or is otherwise free of, bulk ofcellular components or in vitro reaction components such as for example,macromolecules such as lipids or proteins, small biological molecules,and the like.

3. Nucleic Acid Segments

In certain embodiments, the nucleic acid is a nucleic acid segment. Asused herein, the term “nucleic acid segment,” are fragments of a nucleicacid, such as, for a non-limiting example, those that encode only partof an endothelin gene system member locus or gene sequence. Thus, a“nucleic acid segment” may comprise any part of a gene sequence,including from about 2 nucleotides to the full length gene includingpromoter regions to the polyadenylation signal and any length thatincludes all the coding region.

Various nucleic acid segments may be designed based on a particularnucleic acid sequence, and may be of any length. By assigning numericvalues to a sequence, for example, the first residue is 1, the secondresidue is 2, etc., an algorithm defining all nucleic acid segments canbe created:n to n+ywhere n is an integer from 1 to the last number of the sequence and y isthe length of the nucleic acid segment minus one, where n+y does notexceed the last number of the sequence. Thus, for a 10-mer, the nucleicacid segments correspond to bases 1 to 10, 2 to 11, 3 to 12 . . . and soon. For a 15-mer, the nucleic acid segments correspond to bases 1 to 15,2 to 16, 3 to 17 . . . and so on. For a 20-mer, the nucleic segmentscorrespond to bases 1 to 20, 2 to 21, 3 to 22 . . . and so on. Incertain embodiments, the nucleic acid segment may be a probe or primer.As used herein, a “probe” generally refers to a nucleic acid used in adetection method or composition. As used herein, a “primer” generallyrefers to a nucleic acid used in an extension or amplification method orcomposition.

4. Nucleic Acid Complements

The present invention also encompasses a nucleic acid that iscomplementary to a nucleic acid. A nucleic acid is “complement(s)” or is“complementary” to another nucleic acid when it is capable ofbase-pairing with another nucleic acid according to the standardWatson-Crick, Hoogsteen or reverse Hoogsteen binding complementarityrules. As used herein “another nucleic acid” may refer to a separatemolecule or a spatial separated sequence of the same molecule. Inpreferred embodiments, a complement is a hybridization probe oramplification primer for the detection of a nucleic acid polymorphism.

As used herein, the term “complementary” or “complement” also refers toa nucleic acid comprising a sequence of consecutive nucleobases orsemiconsecutive nucleobases (e.g., one or more nucleobase moieties arenot present in the molecule) capable of hybridizing to another nucleicacid strand or duplex even if less than all the nucleobases do not basepair with a counterpart nucleobase. However, in some diagnostic ordetection embodiments, completely complementary nucleic acids arepreferred.

5. Nucleic Acid Detection and Evaluation

Genotyping may be performed using methods as described in Example 1below, or as previously described in Small et al. (2002), which isincorporated herein by reference. It will be understood by the skilledartisan that other standard techniques are available for genotyping andany technique may be used with the present invention. General methods ofnucleic acid detection methods are provided below, followed by specificexamples employed for the identification of polymorphisms, includingsingle nucleotide polymorphisms (SNPs).

Those in the art will readily recognize that nucleic acid molecules maybe double-stranded molecules and that reference to a particular site onone strand refers, as well, to the corresponding site on a complementarystrand. Thus, in defining a polymorphic site, reference to an adenine, athymine (uridine), a cytosine, or a guanine at a particular site on theplus (sense or coding) strand of a nucleic acid molecule is alsointended to include the thymine (uridine), adenine, guanine, or cytosine(respectively) at the corresponding site on a minus (antisense ornoncoding) strand of a complementary strand of a nucleic acid molecule.Thus, reference may be made to either strand and still comprise the samepolymorphic site and an oligonucleotide may be designed to hybridize toeither strand. Throughout the text, in identifying a polymorphic site,reference is made to the sense strand, only for the purpose ofconvenience.

A nucleic acid mixture may be isolated from a biological sample takenfrom the individual, such as a blood sample or tissue sample, or anybiological sample discussed herein, using standard techniques such asdisclosed in Jones (1963) which is hereby incorporated by reference. Thenucleic acid mixture may be comprised of genomic DNA, mRNA, or cDNA and,in the latter two cases, the biological sample must be obtained from anorgan in which an endothelin gene system member is expressed.Furthermore it will be understood by the skilled artisan that mRNA orcDNA preparations would not be used to detect polymorphisms located inintrons or in 5′ and 3′ nontranscribed regions.

The ability to predict a patient's response to a β-adrenergic receptortargeting agent is useful for physicians in making decisions about howto treat a patient having heart failure. A patient whose genotypeindicates the patient will probably respond well to the agent would be abetter candidate for β-adrenergic receptor targeting agent therapy thana patient who is likely to exhibit an intermediate response or noresponse, and the physician would be able to determine with less trialand error which individuals should receive an alternative form oftherapy.

In the genotyping methods used in the present invention, the identity ofa nucleotide (or nucleotide pair) at a polymorphic site may bedetermined by amplifying a target region(s) containing the polymorphicsite(s) directly from one or both copies of a β-adrenergic receptortargeting agent gene present in the individual and the sequence of theamplified region(s) determined by conventional methods. It will bereadily appreciated by the skilled artisan that only one nucleotide willbe detected at a polymorphic site in individuals who are homozygous atthat site, while two different nucleotides will be detected if theindividual is heterozygous for that site. The polymorphism may beidentified directly, known as positive-type identification, or byinference, referred to as negative-type identification. For example,where a SNP is known to be guanine and cytosine in a referencepopulation, a site may be positively determined to be either guanine orcytosine for an individual homozygous at that site, or both guanine andcytosine, if the individual is heterozygous at that site. Alternatively,the site may be negatively determined to be not guanine (and thuscytosine/cytosine) or not cytosine (and thus guanine/guanine).

The target region(s) may be amplified using any oligonucleotide-directedamplification method, including but not limited to polymerase chainreaction (PCR) (U.S. Pat. No. 4,965,188), ligase chain reaction (LCR)(Barany et al., 1991; WO90/01069), and oligonucleotide ligation assay(OLA) (Landegren et al., 1988). Oligonucleotides useful as primers orprobes in such methods should specifically hybridize to a region of thenucleic acid that contains or is adjacent to the polymorphic site.Typically, the oligonucleotides are between 10 and 35 nucleotides inlength and preferably, between 15 and 30 nucleotides in length. Mostpreferably, the oligonucleotides are 20 to 25 nucleotides long. Theexact length of the oligonucleotide will depend on many factors that areroutinely considered and practiced by the skilled artisan.

Other known nucleic acid amplification procedures may be used to amplifythe target region including transcription-based amplification systems(U.S. Pat. No. 5,130,238; EP 329,822; U.S. Pat. No. 5,169,766,WO89/06700) and isothermal methods (Walker et al., 1992).

A polymorphism in the target region may also be assayed before or afteramplification using one of several hybridization-based methods known inthe art. Typically, allele-specific oligonucleotides are utilized inperforming such methods. The allele-specific oligonucleotides may beused as differently labeled probe pairs, with one member of the pairshowing a perfect match to one variant of a target sequence and theother member showing a perfect match to a different variant. In someembodiments, more than one polymorphic site may be detected at onceusing a set of allele-specific oligonucleotides or oligonucleotidepairs.

Hybridization of an allele-specific oligonucleotide to a targetpolynucleotide may be performed with both entities in solution, or suchhybridization may be performed when either the oligonucleotide or thetarget polynucleotide is covalently or noncovalently affixed to a solidsupport. Attachment may be mediated, for example, by antibody-antigeninteractions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges,hydrophobic interactions, chemical linkages, UV cross-linking baking,etc. Allele-specific oligonucleotides may be synthesized directly on thesolid support or attached to the solid support subsequent to synthesis.Solid-supports suitable for use in detection methods of the inventioninclude substrates made of silicon, glass, plastic, paper and the like,which may be formed, for example, into wells (as in 96-well plates),slides, sheets, membranes, fibers, chips, dishes and beads. The solidsupport may be treated, coated or derivatized to facilitate theimmobilization of the allele-specific oligonucleotide or target nucleicacid.

The genotype for one or more polymorphic sites in an endothelin genesystem member of an individual may also be determined by hybridizationof one or both copies of the gene, or a fragment thereof, to nucleicacid arrays and subarrays such as described in WO 95/11995. The arrayswould contain a battery of allele-specific oligonucleotides representingeach of the polymorphic sites to be included in the genotype orhaplotype.

The identity of polymorphisms may also be determined using a mismatchdetection technique, including but not limited to the RNase protectionmethod using riboprobes (Winter et al., 1985; Meyers et al., 1985) andproteins which recognize nucleotide mismatches, such as the E. coli mutSprotein (Modrich, 1991). Alternatively, variant alleles can beidentified by single strand conformation polymorphism (SSCP) analysis(Orita et al., 1989; Humphries et al., 1996) or denaturing gradient gelelectrophoresis (DGGE) (Wartell et al., 1990; Sheffield et al., 1989).

A polymerase-mediated primer extension method may also be used toidentify the polymorphism(s). Several such methods have been describedin the patent and scientific literature. Extended primers containing apolymorphism may be detected by mass spectrometry as described in U.S.Pat. No. 5,605,798. An other primer extension method is allele-specificPCR (Ruano et al., 1989); Ruano et al., 1991; WO 93/22456; Turki et al.,1995).

Polymorphic variations in endothelin gene system members can also bedetected using differential digestion of DNA by certain restrictionenzymes (Small et al., 2002) or by any other method that identifiespolymorphisms.

a. Hybridization

The use of a probe or primer of between 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 60, 70, 80, 90, or 100nucleotides, preferably between 17 and 100 nucleotides in length, or insome aspects of the invention up to 1-2 kilobases or more in length,allows the formation of a duplex molecule that is both stable andselective. Molecules having complementary sequences over contiguousstretches greater than 20 bases in length are generally preferred, toincrease stability and/or selectivity of the hybrid molecules obtained.One will generally prefer to design nucleic acid molecules forhybridization having one or more complementary sequences of 20 to 30nucleotides, or even longer where desired. Such fragments may be readilyprepared, for example, by directly synthesizing the fragment by chemicalmeans or by introducing selected sequences into recombinant vectors forrecombinant production.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNAs and/or RNAs or to provide primers for amplification ofDNA or RNA from samples. Depending on the application envisioned, onewould desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of the probe or primers for the targetsequence.

For applications requiring high selectivity, one will typically desireto employ relatively high stringency conditions to form the hybrids. Forexample, relatively low salt and/or high temperature conditions, such asprovided by about 0.02 M to about 0.10 M NaCl at temperatures of about50° C. to about 70° C. Such high stringency conditions tolerate little,if any, mismatch between the probe or primers and the template or targetstrand and would be particularly suitable for isolating specific genesor for detecting a specific polymorphism. It is generally appreciatedthat conditions can be rendered more stringent by the addition ofincreasing amounts of formamide. For example, under highly stringentconditions, hybridization to filter-bound DNA may be carried out in 0.5M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel et al., 1989).

Conditions may be rendered less stringent by increasing saltconcentration and/or decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25 M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Under lowstringent conditions, such as moderately stringent conditions thewashing may be carried out for example in 0.2×SSC/0.1% SDS at 42° C.(Ausubel et al., 1989). Hybridization conditions can be readilymanipulated depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 1.0 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C.

In certain embodiments, it will be advantageous to employ nucleic acidsof defined sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of being detected. In preferredembodiments, one may desire to employ a fluorescent label or an enzymetag such as urease, alkaline phosphatase or peroxidase, instead ofradioactive or other environmentally undesirable reagents. In the caseof enzyme tags, colorimetric indicator substrates are known that can beemployed to provide a detection means that is visibly orspectrophotometrically detectable, to identify specific hybridizationwith complementary nucleic acid containing samples. In other aspects, aparticular nuclease cleavage site may be present and detection of aparticular nucleotide sequence can be determined by the presence orabsence of nucleic acid cleavage.

In general, it is envisioned that the probes or primers described hereinwill be useful as reagents in solution hybridization, as in PCR, fordetection of expression or genotype of corresponding genes, as well asin embodiments employing a solid phase. In embodiments involving a solidphase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to hybridization with selected probes under desiredconditions. The conditions selected will depend on the particularcircumstances (depending, for example, on the G+C content, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Optimization of hybridization conditions for theparticular application of interest is well known to those of skill inthe art. After washing of the hybridized molecules to removenon-specifically bound probe molecules, hybridization is detected,and/or quantified, by determining the amount of bound label.Representative solid phase hybridization methods are disclosed in U.S.Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods ofhybridization that may be used in the practice of the present inventionare disclosed in U.S. Pat. Nos. 5,849,481, 5,849,486 and 5,851,772. Therelevant portions of these and other references identified in thissection of the Specification are incorporated herein by reference.

b. Amplification of Nucleic Acids

Nucleic acids used as a template for amplification may be isolated fromcells, tissues or other samples according to standard methodologies(Sambrook et al., 2001). In certain embodiments, analysis is performedon whole cell or tissue homogenates or biological fluid samples with orwithout substantial purification of the template nucleic acid. Thenucleic acid may be genomic DNA or fractionated or whole cell RNA. WhereRNA is used, it may be desired to first convert the RNA to acomplementary DNA.

The term “primer,” as used herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty and/or thirty base pairs in length, but longersequences can be employed. Primers may be provided in double-strandedand/or single-stranded form, although the single-stranded form ispreferred.

Pairs of primers designed to selectively hybridize to nucleic acidscorresponding to the endothelin gene system member gene locus, orvariants thereof, and fragments thereof are contacted with the templatenucleic acid under conditions that permit selective hybridization.Depending upon the desired application, high stringency hybridizationconditions may be selected that will only allow hybridization tosequences that are completely complementary to the primers. In otherembodiments, hybridization may occur under reduced stringency to allowfor amplification of nucleic acids that contain one or more mismatcheswith the primer sequences. Once hybridized, the template-primer complexis contacted with one or more enzymes that facilitate template-dependentnucleic acid synthesis. Multiple rounds of amplification, also referredto as “cycles,” are conducted until a sufficient amount of amplificationproduct is produced.

The amplification product may be detected, analyzed or quantified. Incertain applications, the detection may be performed by visual means. Incertain applications, the detection may involve indirect identificationof the product via chemiluminescence, radioactive scintigraphy ofincorporated radiolabel or fluorescent label or even via a system usingelectrical and/or thermal impulse signals (Affymax technology; Bellus,1994).

A number of template dependent processes are available to amplify theoligonucleotide sequences present in a given template sample. One of thebest known amplification methods is the polymerase chain reaction(referred to as PCR™) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1988, each ofwhich is incorporated herein by reference in their entirety.

Another method for amplification is ligase chain reaction (“LCR”),disclosed in European Application No. 320 308, incorporated herein byreference in its entirety. U.S. Pat. No. 4,883,750 describes a methodsimilar to LCR for binding probe pairs to a target sequence. A methodbased on PCR™ and oligonucleotide ligase assay (OLA) (described infurther detail below), disclosed in U.S. Pat. No. 5,912,148, may also beused.

Alternative methods for amplification of target nucleic acid sequencesthat may be used in the practice of the present invention are disclosedin U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497,5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905,5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, Great BritainApplication 2 202 328, and in PCT Application PCT/US89/01025, each ofwhich is incorporated herein by reference in its entirety. QbetaReplicase, described in PCT Application PCT/US87/00880, may also be usedas an amplification method in the present invention.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention (Walker et al., 1992). StrandDisplacement Amplification (SDA), disclosed in U.S. Pat. No. 5,916,779,is another method of carrying out isothermal amplification of nucleicacids which involves multiple rounds of strand displacement andsynthesis, i.e., nick translation

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989; PCT Application WO88/10315, incorporated herein by reference in their entirety). EuropeanApplication 329,822 disclose a nucleic acid amplification processinvolving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA,and double-stranded DNA (dsDNA), which may be used in accordance withthe present invention.

PCT Application WO 89/06700 (incorporated herein by reference in itsentirety) disclose a nucleic acid sequence amplification scheme based onthe hybridization of a promoter region/primer sequence to a targetsingle-stranded DNA (“ssDNA”) followed by transcription of many RNAcopies of the sequence. This scheme is not cyclic, i.e., new templatesare not produced from the resultant RNA transcripts. Other amplificationmethods include “RACE” and “one-sided PCR” (Frohman, 1990; Ohara et al.,1989).

c. Detection of Nucleic Acids

Following any amplification, it may be desirable to separate theamplification product from the template and/or the excess primer. In oneembodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (Sambrook et al., 2001). Separated amplification products may becut out and eluted from the gel for further manipulation. Using lowmelting point agarose gels, the separated band may be removed by heatingthe gel, followed by extraction of the nucleic acid.

Separation of nucleic acids may also be effected by spin columns and/orchromatographic techniques known in art. There are many kinds ofchromatography which may be used in the practice of the presentinvention, including adsorption, partition, ion-exchange,hydroxylapatite, molecular sieve, reverse-phase, column, paper,thin-layer, and gas chromatography as well as HPLC.

In certain embodiments, the amplification products are visualized, withor without separation. A typical visualization method involves stainingof a gel with ethidium bromide and visualization of bands under UVlight. Alternatively, if the amplification products are integrallylabeled with radio- or fluorometrically-labeled nucleotides, theseparated amplification products can be exposed to x-ray film orvisualized under the appropriate excitatory spectra.

In one embodiment, following separation of amplification products, alabeled nucleic acid probe is brought into contact with the amplifiedmarker sequence. The probe preferably is conjugated to a chromophore butmay be radiolabeled. In another embodiment, the probe is conjugated to abinding partner, such as an antibody or biotin, or another bindingpartner carrying a detectable moiety.

In particular embodiments, detection is by Southern blotting andhybridization with a labeled probe. The techniques involved in Southernblotting are well known to those of skill in the art (see Sambrook etal., 2001). One example of the foregoing is described in U.S. Pat. No.5,279,721, incorporated by reference herein, which discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids. The apparatus permits electrophoresis and blottingwithout external manipulation of the gel and is ideally suited tocarrying out methods according to the present invention.

Other methods of nucleic acid detection that may be used in the practiceof the instant invention are disclosed in U.S. Pat. Nos. 5,840,873,5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729,5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, 5,861,244,5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124,5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227,5,932,413 and 5,935,791, each of which is incorporated herein byreference.

d. Other Assays

Other methods for genetic screening may be used within the scope of thepresent invention, for example, to detect mutations in genomic DNA, cDNAand/or RNA samples. Methods used to detect point mutations includedenaturing gradient gel electrophoresis (“DGGE”), restriction fragmentlength polymorphism analysis (“RFLP”), chemical or enzymatic cleavagemethods, direct sequencing of target regions amplified by PCR™ (seeabove), single-strand conformation polymorphism analysis (“SSCP”) andother methods well known in the art.

One method of screening for point mutations is based on RNase cleavageof base pair mismatches in RNA/DNA or RNA/RNA heteroduplexes. As usedherein, the term “mismatch” is defined as a region of one or moreunpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNAor DNA/DNA molecule. This definition thus includes mismatches due toinsertion/deletion mutations, as well as single or multiple base pointmutations.

U.S. Pat. No. 4,946,773 describes an RNase A mismatch cleavage assaythat involves annealing single-stranded DNA or RNA test samples to anRNA probe, and subsequent treatment of the nucleic acid duplexes withRNase A. For the detection of mismatches, the single-stranded productsof the RNase A treatment, electrophoretically separated according tosize, are compared to similarly treated control duplexes. Samplescontaining smaller fragments (cleavage products) not seen in the controlduplex are scored as positive.

Other investigators have described the use of RNase I in mismatchassays. The use of RNase I for mismatch detection is described inliterature from Promega Biotech. Promega markets a kit containing RNaseI that is reported to cleave three out of four known mismatches. Othershave described using the MutS protein or other DNA-repair enzymes fordetection of single-base mismatches.

Alternative methods for detection of deletion, insertion or substitutionmutations that may be used in the practice of the present invention aredisclosed in U.S. Pat. Nos. 5,849,483, 5,851,770, 5,866,337, 5,925,525and 5,928,870, each of which is incorporated herein by reference in itsentirety.

e. Specific Examples of Polymorphism Nucleic Acid Screening Methods

Spontaneous mutations that arise during the course of evolution in thegenomes of organisms are often not immediately transmitted throughoutall of the members of the species, thereby creating polymorphic allelesthat co-exist in the species populations. Often polymorphisms are thecause of genetic diseases. Several classes of polymorphisms have beenidentified. For example, variable nucleotide type polymorphisms (VNTRs),arise from spontaneous tandem duplications of di- or trinucleotiderepeated motifs of nucleotides. If such variations alter the lengths ofDNA fragments generated by restriction endonuclease cleavage, thevariations are referred to as restriction fragment length polymorphisms(RFLPs). RFLPs are been widely used in human and animal geneticanalyses.

Another class of polymorphisms are generated by the replacement of asingle nucleotide. Such single nucleotide polymorphisms (SNPs) rarelyresult in changes in a restriction endonuclease site. Thus, SNPs arerarely detectable restriction fragment length analysis. SNPs are themost common genetic variations and occur once every 100 to 300 bases andseveral SNP mutations have been found that affect a single nucleotide ina protein-encoding gene in a manner sufficient to actually cause agenetic disease. SNP diseases are exemplified by hemophilia, sickle-cellanemia, hereditary hemochromatosis, late-onset Alzheimer's disease etc.

Several methods have been developed to screen polymorphisms and someexamples are listed below. The reference of Kwok and Chen (2003) andKwok (2001) provide overviews of some of these methods; both of thesereferences are specifically incorporated by reference.

SNPs relating to endothelin gene system members can be characterized bythe use of any of these methods or suitable modification thereof. Suchmethods include the direct or indirect sequencing of the site, the useof restriction enzymes where the respective alleles of the site createor destroy a restriction site, the use of allele-specific hybridizationprobes, the use of antibodies that are specific for the proteins encodedby the different alleles of the polymorphism, or any other biochemicalinterpretation.

i. DNA Sequencing

The most commonly used method of characterizing a polymorphism is directDNA sequencing of the genetic locus that flanks and includes thepolymorphism. Such analysis can be accomplished using either the“dideoxy-mediated chain termination method,” also known as the “SangerMethod” (Sanger et al., 1975) or the “chemical degradation method,” alsoknown as the “Maxam-Gilbert method” (Maxam et al., 1977). Sequencing incombination with genomic sequence-specific amplification technologies,such as the polymerase chain reaction may be utilized to facilitate therecovery of the desired genes (Mullis et al., 1986; European PatentApplication 50,424; European Patent Application. 84,796, European PatentApplication 258,017, European Patent Application. 237,362; EuropeanPatent Application. 201,184; U.S. Pat. Nos. 4,683,202; 4,582,788; and4,683,194), all of the above incorporated herein by reference.

ii. Exonuclease Resistance

Other methods that can be employed to determine the identity of anucleotide present at a polymorphic site utilize a specializedexonuclease-resistant nucleotide derivative (U.S. Pat. No. 4,656,127). Aprimer complementary to an allelic sequence immediately 3′- to thepolymorphic site is hybridized to the DNA under investigation. If thepolymorphic site on the DNA contains a nucleotide that is complementaryto the particular exonucleotide-resistant nucleotide derivative present,then that derivative will be incorporated by a polymerase onto the endof the hybridized primer. Such incorporation makes the primer resistantto exonuclease cleavage and thereby permits its detection. As theidentity of the exonucleotide-resistant derivative is known one candetermine the specific nucleotide present in the polymorphic site of theDNA.

iii. Microsequencing Methods

Several other primer-guided nucleotide incorporation procedures forassaying polymorphic sites in DNA have been described (Komher et al.,1989; Sokolov, 1990; Syvanen 1990; Kuppuswamy et al., 1991; Prezant etal., 1992; Ugozzoll et al., 1992; Nyren et al., 1993). These methodsrely on the incorporation of labeled deoxynucleotides to discriminatebetween bases at a polymorphic site. As the signal is proportional tothe number of deoxynucleotides incorporated, polymorphisms that occur inruns of the same nucleotide result in a signal that is proportional tothe length of the run (Syvanen et al., 1990).

iv. Extension in Solution

French Patent 2,650,840 and PCT Application WO91/02087 discuss asolution-based method for determining the identity of the nucleotide ofa polymorphic site. According to these methods, a primer complementaryto allelic sequences immediately 3′- to a polymorphic site is used. Theidentity of the nucleotide of that site is determined using labeleddideoxynucleotide derivatives which are incorporated at the end of theprimer if complementary to the nucleotide of the polymorphic site.

v. Genetic Bit Analysis or Solid-Phase Extension

PCT Application WO92/15712 describes a method that uses mixtures oflabeled terminators and a primer that is complementary to the sequence3′ to a polymorphic site. The labeled terminator that is incorporated iscomplementary to the nucleotide present in the polymorphic site of thetarget molecule being evaluated and is thus identified. Here the primeror the target molecule is immobilized to a solid phase.

vi. Oligonucleotide Ligation Assay (OLA)

This is another solid phase method that uses different methodology(Landegren et al., 1988). Two oligonucleotides, capable of hybridizingto abutting sequences of a single strand of a target DNA are used. Oneof these oligonucleotides is biotinylated while the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation permits the recovery ofthe labeled oligonucleotide by using avidin. Other nucleic aciddetection assays, based on this method, combined with PCR have also beendescribed (Nickerson et al., 1990). Here PCR is used to achieve theexponential amplification of target DNA, which is then detected usingthe OLA.

vii. Ligase/Polymerase-Mediated Genetic Bit Analysis

U.S. Pat. No. 5,952,174 describes a method that also involves twoprimers capable of hybridizing to abutting sequences of a targetmolecule. The hybridized product is formed on a solid support to whichthe target is immobilized. Here the hybridization occurs such that theprimers are separated from one another by a space of a singlenucleotide. Incubating this hybridized product in the presence of apolymerase, a ligase, and a nucleoside triphosphate mixture containingat least one deoxynucleoside triphosphate allows the ligation of anypair of abutting hybridized oligonucleotides. Addition of a ligaseresults in two events required to generate a signal, extension andligation. This provides a higher specificity and lower “noise” thanmethods using either extension or ligation alone and unlike thepolymerase-based assays, this method enhances the specificity of thepolymerase step by combining it with a second hybridization and aligation step for a signal to be attached to the solid phase.

viii. Invasive Cleavage Reactions

Invasive cleavage reactions can be used to evaluate cellular DNA for aparticular polymorphism. A technology called INVADER® employs suchreactions (e.g., de Arruda et al., 2002; Stevens et al., 2003, which areincorporated by reference). Generally, there are three nucleic acidmolecules: 1) an oligonucleotide upstream of the target site (“upstreamoligo”), 2) a probe oligonucleotide covering the target site (“probe”),and 3) a single-stranded DNA with the target site (“target”). Theupstream oligo and probe do not overlap but they contain contiguoussequences. The probe contains a donor fluorophore, such as fluoroscein,and an acceptor dye, such as Dabcyl. The nucleotide at the 3′ terminalend of the upstream oligo overlaps (“invades”) the first base pair of aprobe-target duplex. Then the probe is cleaved by a structure-specific5′ nuclease causing separation of the fluorophore/quencher pair, whichincreases the amount of fluorescence that can be detected. See Lu etal., 2004.

In some cases, the assay is conducted on a solid-surface or in an arrayformat.

ix. Other Methods to Detect SNPs

Several other specific methods for polymorphism detection andidentification are presented below and may be used as such or withsuitable modifications in conjunction with identifying polymorphisms ofendothelin gene system members in the present invention. Several othermethods are also described on the SNP web site of the NCBI on the worldwide web at ncbi.nlm.nih.gov/SNP, incorporated herein by reference.

In a particular embodiment, extended haplotypes may be determined at anygiven locus in a population, which allows one to identify exactly whichSNPs will be redundant and which will be essential in associationstudies. The latter is referred to as “haplotype tag SNPs (htSNPs),”markers that capture the haplotypes of a gene or a region of linkagedisequilibrium. See Johnson et al. (2001) and Ke and Cardon (2003), eachof which is incorporated herein by reference, for exemplary methods.

The VDA-assay utilizes PCR amplification of genomic segments by long PCRmethods using TaKaRa LA Taq reagents and other standard reactionconditions. The long amplification can amplify DNA sizes of about2,000-12,000 bp. Hybridization of products to variant detector array(VDA) can be performed by a Affymetrix High Throughput Screening Centerand analyzed with computerized software.

A method called Chip Assay uses PCR amplification of genomic segments bystandard or long PCR protocols. Hybridization products are analyzed byVDA, Halushka et al. (1999), incorporated herein by reference. SNPs aregenerally classified as “Certain” or “Likely” based on computer analysisof hybridization patterns. By comparison to alternative detectionmethods such as nucleotide sequencing, “Certain” SNPs have beenconfirmed 100% of the time; and “Likely” SNPs have been confirmed 73% ofthe time by this method.

Other methods simply involve PCR amplification following digestion withthe relevant restriction enzyme. Yet others involve sequencing ofpurified PCR products from known genomic regions.

In yet another method, individual exons or overlapping fragments oflarge exons are PCR-amplified. Primers are designed from published ordatabase sequences and PCR-amplification of genomic DNA is performedusing the following conditions: 200 ng DNA template, 0.5 μM each primer,80 μM each of dCTP, dATP, dTTP and dGTP, 5% formamide, 1.5 mM MgCl₂, 0.5U of Taq polymerase and 0.1 volume of the Taq buffer. Thermal cycling isperformed and resulting PCR-products are analyzed by PCR-single strandconformation polymorphism (PCR-SSCP) analysis, under a variety ofconditions, e.g, 5 or 10% polyacrylamide gel with 15% urea, with orwithout 5% glycerol. Electrophoresis is performed overnight.PCR-products that show mobility shifts are reamplified and sequenced toidentify nucleotide variation.

In a method called CGAP-GAI (DEMIGLACE), sequence and alignment data(from a PHRAP.ace file), quality scores for the sequence base calls(from PHRED quality files), distance information (from PHYLIP dnadistand neighbour programs) and base-calling data (from PHRED “-d” switch)are loaded into memory. Sequences are aligned and examined for eachvertical chunk (‘slice’) of the resulting assembly for disagreement. Anysuch slice is considered a candidate SNP (DEMIGLACE). A number offilters are used by DEMIGLACE to eliminate slices that are not likely torepresent true polymorphisms. These include filters that: (i) excludesequences in any given slice from SNP consideration where neighboringsequence quality scores drop 40% or more; (ii) exclude calls in whichpeak amplitude is below the fifteenth percentile of all base calls forthat nucleotide type; (iii) disqualify regions of a sequence having ahigh number of disagreements with the consensus from participating inSNP calculations; (iv) removed from consideration any base call with analternative call in which the peak takes up 25% or more of the area ofthe called peak; (v) exclude variations that occur in only one readdirection. PHRED quality scores were converted into probability-of-errorvalues for each nucleotide in the slice. Standard Baysian methods areused to calculate the posterior probability that there is evidence ofnucleotide heterogeneity at a given location.

In a method called CU-RDF (RESEQ), PCR amplification is performed fromDNA isolated from blood using specific primers for each SNP, and aftertypical cleanup protocols to remove unused primers and free nucleotides,direct sequencing using the same or nested primers.

In a method called DEBNICK (METHOD-B), a comparative analysis ofclustered EST sequences is performed and confirmed by fluorescent-basedDNA sequencing. In a related method, called DEBNICK (METHOD-C),comparative analysis of clustered EST sequences with phred quality >20at the site of the mismatch, average phred quality >=20 over 5 bases5′-FLANK and 3′ to the SNP, no mismatches in 5 bases 5′ and 3′ to theSNP, at least two occurrences of each allele is performed and confirmedby examining traces.

In a method identified by ERO (RESEQ), new primers sets are designed forelectronically published STSs and used to amplify DNA from 10 differentmouse strains. The amplification product from each strain is then gelpurified and sequenced using a standard dideoxy, cycle sequencingtechnique with ³³P-labeled terminators. All the ddATP terminatedreactions are then loaded in adjacent lanes of a sequencing gel followedby all of the ddGTP reactions and so on. SNPs are identified by visuallyscanning the radiographs.

In another method identified as ERO (RESEQ-HT), new primers sets aredesigned for electronically published murine DNA sequences and used toamplify DNA from 10 different mouse strains. The amplification productfrom each strain is prepared for sequencing by treating with ExonucleaseI and Shrimp Alkaline Phosphatase. Sequencing is performed using ABIPrism Big Dye Terminator Ready Reaction Kit (Perkin-Elmer) and sequencesamples are run on the 3700 DNA Analyzer (96 Capillary Sequencer).

FGU-CBT (SCA2-SNP) identifies a method where the region containing theSNP were PCR amplified using the primers SCA2-FP3 and SCA2-RP3.Approximately 100 ng of genomic DNA is amplified in a 50 ml reactionvolume containing a final concentration of 5 mM Tris, 25 mM KCl, 0.75 mMMgCl₂, 0.05% gelatin, 20 pmol of each primer and 0.5 U of Taq DNApolymerase. Samples are denatured, annealed and extended and the PCRproduct is purified from a band cut out of the agarose gel using, forexample, the QIAquick gel extraction kit (Qiagen) and is sequenced usingdye terminator chemistry on an ABI Prism 377 automated DNA sequencerwith the PCR primers.

In a method identified as JBLACK (SEQ/RESTRICT), two independent PCRreactions are performed with genomic DNA. Products from the firstreaction are analyzed by sequencing, indicating a unique FspIrestriction site. The mutation is confirmed in the product of the secondPCR reaction by digesting with Fsp I.

In a method described as KWOK(1), SNPs are identified by comparing highquality genomic sequence data from four randomly chosen individuals bydirect DNA sequencing of PCR products with dye-terminator chemistry (seeKwok et al., 1996). In a related method identified as KWOK(2) SNPs areidentified by comparing high quality genomic sequence data fromoverlapping large-insert clones such as bacterial artificial chromosomes(BACs) or P1-based artificial chromosomes (PACs). An STS containing thisSNP is then developed and the existence of the SNP in variouspopulations is confirmed by pooled DNA sequencing (see Taillon-Miller etal., 1998). In another similar method called KWOK(3), SNPs areidentified by comparing high quality genomic sequence data fromoverlapping large-insert clones BACs or PACs. The SNPs found by thisapproach represent DNA sequence variations between the two donorchromosomes but the allele frequencies in the general population havenot yet been determined. In method KWOK(5), SNPs are identified bycomparing high quality genomic sequence data from a homozygous DNAsample and one or more pooled DNA samples by direct DNA sequencing ofPCR products with dye-terminator chemistry. The STSs used are developedfrom sequence data found in publicly available databases. Specifically,these STSs are amplified by PCR against a complete hydatidiform mole(CHM) that has been shown to be homozygous at all loci and a pool of DNAsamples from 80 CEPH parents (see Kwok et al., 1994).

In another such method, KWOK (OverlapSnpDetectionWithPolyBayes), SNPsare discovered by automated computer analysis of overlapping regions oflarge-insert human genomic clone sequences. For data acquisition, clonesequences are obtained directly from large-scale sequencing centers.This is necessary because base quality sequences are notpresent/available through GenBank. Raw data processing involves analyzedof clone sequences and accompanying base quality information forconsistency. Finished (‘base perfect’, error rate lower than 1 in 10,000bp) sequences with no associated base quality sequences are assigned auniform base quality value of 40 (1 in 10,000 bp error rate). Draftsequences without base quality values are rejected. Processed sequencesare entered into a local database. A version of each sequence with knownhuman repeats masked is also stored. Repeat masking is performed withthe program “MASKERAID.” Overlap detection: Putative overlaps aredetected with the program “WUBLAST.” Several filtering steps followed inorder to eliminate false overlap detection results, i.e. similaritiesbetween a pair of clone sequences that arise due to sequence duplicationas opposed to true overlap. Total length of overlap, overall percentsimilarity, number of sequence differences between nucleotides with highbase quality value “high-quality mismatches.” Results are also comparedto results of restriction fragment mapping of genomic clones atWashington University Genome Sequencing Center, finisher's reports onoverlaps, and results of the sequence contig building effort at theNCBI. SNP detection: Overlapping pairs of clone sequence are analyzedfor candidate SNP sites with the ‘POLYBAYES’ SNP detection software.Sequence differences between the pair of sequences are scored for theprobability of representing true sequence variation as opposed tosequencing error. This process requires the presence of base qualityvalues for both sequences. High-scoring candidates are extracted. Thesearch is restricted to substitution-type single base pair variations.Confidence score of candidate SNP is computed by the POLYBAYES software.

In method identified by KWOK (TaqMan assay), the TaqMan assay is used todetermine genotypes for 90 random individuals. In method identified byKYUGEN(Q1), DNA samples of indicated populations are pooled and analyzedby PLACE-SSCP. Peak heights of each allele in the pooled analysis arecorrected by those in a heterozygote, and are subsequently used forcalculation of allele frequencies. Allele frequencies higher than 10%are reliably quantified by this method. Allele frequency=0 (zero) meansthat the allele was found among individuals, but the corresponding peakis not seen in the examination of pool. Allele frequency=0-0.1 indicatesthat minor alleles are detected in the pool but the peaks are too low toreliably quantify.

In yet another method identified as KYUGEN (Method 1), PCR products arepost-labeled with fluorescent dyes and analyzed by an automatedcapillary electrophoresis system under SSCP conditions (PLACE-SSCP).Four or more individual DNAs are analyzed with or without two pooled DNA(Japanese pool and CEPH parents pool) in a series of experiments.Alleles are identified by visual inspection. Individual DNAs withdifferent genotypes are sequenced and SNPs identified. Allelefrequencies are estimated from peak heights in the pooled samples aftercorrection of signal bias using peak heights in heterozygotes. For thePCR primers are tagged to have 5′-ATT or 5′-GTT at their ends forpost-labeling of both strands. Samples of DNA (10 ng/ul) are amplifiedin reaction mixtures containing the buffer (10 mM Tris-HCl, pH 8.3 or9.3, 50 mM KCl, 2.0 mM MgCl₂), 0.25 μM of each primer, 200 μM of eachdNTP, and 0.025 units/μl of Taq DNA polymerase premixed with anti-Taqantibody. The two strands of PCR products are differentially labeledwith nucleotides modified with R110 and R6G by an exchange reaction ofKlenow fragment of DNA polymerase I. The reaction is stopped by addingEDTA, and unincorporated nucleotides are dephosphorylated by adding calfintestinal alkaline phosphatase. For the SSCP: an aliquot offluorescently labeled PCR products and TAMRA-labeled internal markersare added to deionized formamide, and denatured. Electrophoresis isperformed in a capillary using an ABI Prism 310 Genetic Analyzer.Genescan softwares (P-E Biosystems) are used for data collection anddata processing. DNA of individuals (two to eleven) including those whoshowed different genotypes on SSCP are subjected for direct sequencingusing big-dye terminator chemistry, on ABI Prism 310 sequencers.Multiple sequence trace files obtained from ABI Prism 310 are processedand aligned by Phred/Phrap and viewed using Consed viewer. SNPs areidentified by PolyPhred software and visual inspection.

In yet another method identified as KYUGEN (Method 2), individuals withdifferent genotypes are searched by denaturing HPLC (DHPLC) orPLACE-SSCP (Inazuka et al., 1997) and their sequences are determined toidentify SNPs. PCR is performed with primers tagged with 5′-ATT or5′-GTT at their ends for post-labeling of both strands. DHPLC analysisis carried out using the WAVE DNA fragment analysis system(Transgenomic). PCR products are injected into DNASep column, andseparated under the conditions determined using WAVEMaker program(Transgenomic). The two strands of PCR products that are differentiallylabeled with nucleotides modified with R110 and R6G by an exchangereaction of Klenow fragment of DNA polymerase I. The reaction is stoppedby adding EDTA, and unincorporated nucleotides are dephosphorylated byadding calf intestinal alkaline phosphatase. SSCP followed byelectrophoresis is performed in a capillary using an ABI Prism 310Genetic Analyzer. Genescan softwares (P-E Biosystems). DNA ofindividuals including those who showed different genotypes on DHPLC orSSCP are subjected for direct sequencing using big-dye terminatorchemistry, on ABI Prism 310 sequencer. Multiple sequence trace filesobtained from ABI Prism 310 are processed and aligned by Phred/Phrap andviewed using Consed viewer. SNPs are identified by PolyPhred softwareand visual inspection. Trace chromatogram data of EST sequences inUnigene are processed with PHRED. To identify likely SNPs, single basemismatches are reported from multiple sequence alignments produced bythe programs PHRAP, BRO and POA for each Unigene cluster. BRO correctedpossible misreported EST orientations, while POA identified and analyzednon-linear alignment structures indicative of gene mixing/chimeras thatmight produce spurious SNPs. Bayesian inference is used to weighevidence for true polymorphism versus sequencing error, misalignment orambiguity, misclustering or chimeric EST sequences, assessing data suchas raw chromatogram height, sharpness, overlap and spacing; sequencingerror rates; context-sensitivity; cDNA library origin, etc.

In method identified as MARSHFIELD (Method-B), overlapping human DNAsequences which contained putative insertion/deletion polymorphisms areidentified through searches of public databases. PCR primers whichflanked each polymorphic site are selected from the consensus sequences.Primers are used to amplify individual or pooled human genomic DNA.Resulting PCR products are resolved on a denaturing polyacrylamide geland a PhosphorImager is used to estimate allele frequencies from DNApools.

f. Linkage Disequilibrium

Polymorphisms in linkage disequilibrium with another polymorphism inwhich identification of one polymorphism is predictive of the identityof the linked polymorphism. “Linkage disequilibrium” (“LD” as usedherein, though also referred to as “LED” in the art) refers to asituation where a particular combination of alleles (i.e., a variantform of a given gene) or polymorphisms at two loci appears morefrequently than would be expected by chance. “Significant” as used inrespect to linkage disequilibrium, as determined by one of skill in theart, is contemplated to be a statistical p or α value that may be 0.25or 0.1 and may be 0.1, 0.05, 0.001, 0.00001, or less. The polymorphismat position 198 of EDN1 protein may be determined by evaluating apolymorphism in linkage disequilibrium therewith. The invention may beimplemented in this manner with respect to one or more polymorphisms soas to allow haplotype analysis. “Haplotype” is used according to itsplain and ordinary meaning to one skilled in the art. It refers to acollective genotype of two or more alleles or polymorphisms along one ofthe homologous chromosomes.

g. Pyrosequencing

Pyrosequencing is a method of DNA sequencing based on a “sequencing bysynthesis” principle. Polymorphisms may be detected usingpyrosequencing, and certain embodiments of the present invention employthis method. The method is based on a chemical light-producing enzymaticreaction that is triggered when a molecular recognition event occurs.Simply put, the method allows sequencing of a single strand of DNA bysynthesizing the complementary strand along it. Each time a nucleotideis incorporated into the growing chain, a cascade of enzymatic reactionsis triggered which results in a light signal.

More specifically, pyrosequencing is based on the detection of inorganicpyrophosphates (PPi) released during a polymerase reaction. A sequencingprimer is first hybridized to a single stranded DNA template andincubated with a DNA polymerase. In addition to the polymerase, theenzymes ATP sulfurylase, luciferase, and apyrase are added to thereaction along with the substrates, adenine 5′ phosphosulfate (APS) andluciferin. Subsequently, individual nucleotides are added. When an addednucleotide is complementary to the next available base in the templatestrand, it is incorporated into the extension product, releasingpyrophosphate. In the presence of adenosine 5′ phorphosulfate,pyrophosphate is converted into ATP by apryase in a quantity equimolarto the amount of incorporated nucleotide. The ATP generated by thereaction with apyrase then drives the luciferase-mediated conversion ofluciferin to oxyluciferin, generating visible light in amounts that areproportional to the amount of ATP, and thus the number of nucleotidesincorporated into the growing DNA template. The light produced by theluciferase-catalyzed reaction may be detected by, for example, a chargecoupled device (CCD) camera.

B. Evaluating the Protein

Alternatively, polymorphic variation can be determined by any methodthat detects an amino acid variation at a particular position, such asat position 198 of EDN1 protein. The invention should not be limited byany particular method for achieving this. For example, a sample of fluidor tissue may be obtained from an individual and the amino acid atposition 198 of EDN1 protein is determined. Such detection can be byvarious methods including antibody based assays (Western blots, ELISA)or amino acid analysis (high pressure liquid chromatography or massspectroscopy) could be used that would detect whether the protein hasLys or Asn.

Therefore, in certain embodiments, the present invention concernscompositions comprising at least one proteinaceous molecule, such as aprotein encoded by an endothelin gene system member, or an protein thatbinds thereto, such as an antibody. As used herein, a “proteinaceousmolecule,” “proteinaceous composition,” “proteinaceous compound,”“proteinaceous chain” or “proteinaceous material” generally refers, butis not limited to, a protein of greater than about 200 amino acids orthe full length endogenous sequence translated from a gene; apolypeptide of greater than about 100 amino acids; and/or a peptide offrom about 3 to about 100 amino acids. All the “proteinaceous” termsdescribed above may be used interchangeably herein.

Proteinaceous compositions may be made by any technique known to thoseof skill in the art, including the expression of proteins, polypeptidesor peptides through standard molecular biological techniques, theisolation of proteinaceous compounds from natural sources, or thechemical synthesis of proteinaceous materials. The nucleotide andprotein, polypeptide and peptide sequences for various genes have beenpreviously disclosed, and may be found at computerized databases knownto those of ordinary skill in the art. One such database is the NationalCenter for Biotechnology Information's Genbank and GenPept databases(world wide web at .ncbi.nlm.nih.gov/). The coding regions for theseknown genes may be amplified and/or expressed using the techniquesdisclosed herein or as would be know to those of ordinary skill in theart. Alternatively, various commercial preparations of proteins,polypeptides and peptides are known to those of skill in the art.

1. Protein Purification

It may be desirable to purify an endothelin gene system member from asample or purify a protein that binds endothelin gene system member,such as an antibody. Such techniques are widely employed and theinvention is not intended to be limited with respect to proteinpurification. Protein purification techniques are well known to those ofskill in the art. These techniques involve, at one level, the crudefractionation of the cellular milieu to polypeptide and non-polypeptidefractions. Having separated the polypeptide from other proteins, thepolypeptide of interest may be further purified using chromatographicand electrophoretic techniques to achieve partial or completepurification (or purification to homogeneity). Analytical methodsparticularly suited to the preparation of a pure peptide areion-exchange chromatography, exclusion chromatography; polyacrylamidegel electrophoresis; isoelectric focusing. A particularly efficientmethod of purifying peptides is fast protein liquid chromatography oreven HPLC.

Certain aspects of the present invention may concern the purification,and in particular embodiments, the substantial purification, of anencoded protein or peptide. The term “purified protein or peptide” asused herein, is intended to refer to a composition, isolatable fromother components, wherein the protein or peptide is purified to anydegree relative to its naturally-obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

A variety of techniques suitable for use in protein purification will bewell known to those of skill in the art. These include, for example,precipitation with ammonium sulfate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (e.g., alter pH, ionic strength, and temperature).

A particular type of affinity chromatography useful in the purificationof carbohydrate containing compounds is lectin affinity chromatography.Lectins are a class of substances that bind to a variety ofpolysaccharides and glycoproteins. Lectins are usually coupled toagarose by cyanogen bromide. Conconavalin A coupled to Sepharose was thefirst material of this sort to be used and has been widely used in theisolation of polysaccharides and glycoproteins other lectins that havebeen include lentil lectin, wheat germ agglutinin which has been usefulin the purification of N-acetyl glucosaminyl residues and Helix pomatialectin. Lectins themselves are purified using affinity chromatographywith carbohydrate ligands. Lactose has been used to purify lectins fromcastor bean and peanuts; maltose has been useful in extracting lectinsfrom lentils and jack bean; N-acetyl-D galactosamine is used forpurifying lectins from soybean; N-acetyl glucosaminyl binds to lectinsfrom wheat germ; D-galactosamine has been used in obtaining lectins fromclaims and L-fucose will bind to lectins from lotus.

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand also shouldprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

2. Antibodies

Another embodiment of the present invention are antibodies, in somecases, a human monoclonal antibody immunoreactive with the polypeptidesequence of an endothelin gene system member. It is understood thatantibodies can be used for detecting an endothelin gene system member,particularly an endothelin gene system member that is the result of aparticular polymorphism. It is contemplated that antibodies particularlyuseful in the context of the present invention are those thatdifferentially bind EDN1 protein protein with a lysine or an asparagineat amino acid 198 so as to distinguish between the two populations.

As used herein, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally,IgG and/or IgM are preferred because they are the most common antibodiesin the physiological situation and because they are most easily made ina laboratory setting.

The term “antibody” is used to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)₂, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. The techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (see, e.g., Harlow and Lane, 1988; incorporated herein byreference).

a. Antibody Generation

In certain embodiments, the present invention involves antibodies. Forexample, all or part of a monoclonal may be used in determining theamino acid at position 389. As detailed above, in addition to antibodiesgenerated against full length proteins, antibodies also may be generatedin response to smaller constructs comprising epitopic core regions,including wild-type and mutant epitopes. The techniques for preparingand using various antibody-based constructs and fragments are well knownin the art. Means for preparing and characterizing antibodies are alsowell known in the art (See, e.g., Harlow and Lane, 1988; incorporatedherein by reference).

Monoclonal antibodies (mAbs) are recognized to have certain advantages,e.g., reproducibility and large-scale production, and their use isgenerally preferred. The invention thus provides monoclonal antibodiesof the human, murine, monkey, rat, hamster, rabbit and even chickenorigin.

The methods for generating monoclonal antibodies (mAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody may be prepared by immunizing an animalwith an immunogenic polypeptide composition in accordance with thepresent invention and collecting antisera from that immunized animal.Alternatively, in some embodiments of the present invention, serum iscollected from persons who may have been exposed to a particularantigen. Exposure to a particular antigen may occur a work environment,such that those persons have been occupationally exposed to a particularantigen and have developed polyclonal antibodies to a peptide,polypeptide, or protein. In some embodiments of the invention polyclonalserum from occupationally exposed persons is used to identify antigenicregions in the gelonin toxin through the use of immunodetection methods.

A wide range of animal species can be used for the production ofantisera. Typically the animal used for production of antisera is arabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because ofthe relatively large blood volume of rabbits, a rabbit is a preferredchoice for production of polyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin also canbe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitablemolecule adjuvants include all acceptable immunostimulatory compounds,such as cytokines, toxins or synthetic compositions.

Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12,γ-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such asthur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A(MPL). RIBI, which contains three components extracted from bacteria,MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2%squalene/Tween 80 emulsion also is contemplated. MHC antigens may evenbe used. Exemplary, often preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

In addition to adjuvants, it may be desirable to coadminister biologicresponse modifiers (BRM), which have been shown to upregulate T cellimmunity or downregulate suppressor cell activity. Such BRMs include,but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, Pa.);low-dose Cyclophosphamide (CYP; 300 mg/m²) (Johnson/Mead, N.J.),cytokines such as γ-interferon, IL-2, or IL-12 or genes encodingproteins involved in immune helper functions, such as B-7.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization.

A second, booster injection also may be given. The process of boostingand titering is repeated until a suitable titer is achieved. When adesired level of immunogenicity is obtained, the immunized animal can bebled and the serum isolated and stored, and/or the animal can be used togenerate mAbs.

mAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified polypeptide, peptide or domain, be it a wild-type ormutant composition. The immunizing composition is administered in amanner effective to stimulate antibody producing cells.

mAbs may be further purified, if desired, using filtration,centrifugation and various chromatographic methods such as HPLC oraffinity chromatography. Fragments of the monoclonal antibodies of theinvention can be obtained from the monoclonal antibodies so produced bymethods which include digestion with enzymes, such as pepsin or papain,and/or by cleavage of disulfide bonds by chemical reduction.Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer.

It also is contemplated that a molecular cloning approach may be used togenerate mAbs. For this, combinatorial immunoglobulin phagemid librariesare prepared from RNA isolated from the spleen of the immunized animal,and phagemids expressing appropriate antibodies are selected by panningusing cells expressing the antigen and control cells. The advantages ofthis approach over conventional hybridoma techniques are thatapproximately 10⁴ times as many antibodies can be produced and screenedin a single round, and that new specificities are generated by H and Lchain combination which further increases the chance of findingappropriate antibodies.

b. Immunodetection Methods

As discussed, in some embodiments, the present invention concernsimmunodetection methods for binding, purifying, removing, determining,and/or otherwise detecting biological components such as antigenicregions on polypeptides and peptides. The immunodetection methods of thepresent invention can be used to identify antigenic regions of apeptide, polypeptide, or protein that has therapeutic implications,particularly in reducing the immunogenicity or antigenicity of thepeptide, polypeptide, or protein in a target subject.

Immunodetection methods include enzyme linked immunosorbent assay(ELISA), radioimmunoassay (RIA), immunoradiometric assay,fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, andWestern blot, though several others are well known to those of ordinaryskill. The steps of various useful immunodetection methods have beendescribed in the scientific literature, such as, e.g., Doolittle et al.,1999; Gulbis et al., 1993; De Jager et al., 1993; and Nakamura et al.,1987, each incorporated herein by reference.

In general, the immunobinding methods include obtaining a samplesuspected of containing a protein, polypeptide and/or peptide, andcontacting the sample with a first antibody, monoclonal or polyclonal,in accordance with the present invention, as the case may be, underconditions effective to allow the formation of immunocomplexes.

These methods include methods for purifying a protein, polypeptideand/or peptide from organelle, cell, tissue or organism's samples. Inthese instances, the antibody removes the antigenic protein, polypeptideand/or peptide component from a sample. The antibody will preferably belinked to a solid support, such as in the form of a column matrix, andthe sample suspected of containing the protein, polypeptide and/orpeptide antigenic component will be applied to the immobilized antibody.The unwanted components will be washed from the column, leaving theantigen immunocomplexed to the immobilized antibody to be eluted.

The immunobinding methods also include methods for detecting andquantifying the amount of an antigen component in a sample and thedetection and quantification of any immune complexes formed during thebinding process. Here, one would obtain a sample suspected of containingan antigen or antigenic domain, and contact the sample with an antibodyagainst the antigen or antigenic domain, and then detect and quantifythe amount of immune complexes formed under the specific conditions.

In terms of antigen detection, the biological sample analyzed may be anysample that is suspected of containing an antigen or antigenic domain,such as, for example, a tissue section or specimen, a homogenized tissueextract, a cell, an organelle, separated and/or purified forms of any ofthe above antigen-containing compositions, or even any biological fluidthat comes into contact with the cell or tissue, including blood and/orserum.

Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, any antigenspresent. After this time, the sample-antibody composition, such as atissue section, ELISA plate, dot blot or western blot, will generally bewashed to remove any non-specifically bound antibody species, allowingonly those antibodies specifically bound within the primary immunecomplexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

The antibody employed in the detection may itself be linked to adetectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary immune complexes in thecomposition to be determined. Alternatively, the first antibody thatbecomes bound within the primary immune complexes may be detected bymeans of a second binding ligand that has binding affinity for theantibody. In these cases, the second binding ligand may be linked to adetectable label. The second binding ligand is itself often an antibody,which may thus be termed a “secondary” antibody. The primary immunecomplexes are contacted with the labeled, secondary binding ligand, orantibody, under effective conditions and for a period of time sufficientto allow the formation of secondary immune complexes. The secondaryimmune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the antibody is used to form secondary immunecomplexes, as described above. After washing, the secondary immunecomplexes are contacted with a third binding ligand or antibody that hasbinding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

One method of immunodetection designed by Charles Cantor uses twodifferent antibodies. A first step biotinylated, monoclonal orpolyclonal antibody is used to detect the target antigen(s), and asecond step antibody is then used to detect the biotin attached to thecomplexed biotin. In that method the sample to be tested is firstincubated in a solution containing the first step antibody. If thetarget antigen is present, some of the antibody binds to the antigen toform a biotinylated antibody/antigen complex. The antibody/antigencomplex is then amplified by incubation in successive solutions ofstreptavidin (or avidin), biotinylated DNA, and/or complementarybiotinylated DNA, with each step adding additional biotin sites to theantibody/antigen complex. The amplification steps are repeated until asuitable level of amplification is achieved, at which point the sampleis incubated in a solution containing the second step antibody againstbiotin. This second step antibody is labeled, as for example with anenzyme that can be used to detect the presence of the antibody/antigencomplex by histoenzymology using a chromogen substrate. With suitableamplification, a conjugate can be produced which is macroscopicallyvisible.

Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. At leastin theory, the enormous amplification capability and specificity of PCRcan be utilized to detect a single antigen molecule.

i. ELISAs

As detailed above, immunoassays, in their most simple and/or directsense, are binding assays. Certain preferred immunoassays are thevarious types of enzyme linked immunosorbent assays (ELISAs) and/orradioimmunoassays (RIA) known in the art. Immunohistochemical detectionusing tissue sections is also particularly useful. However, it will bereadily appreciated that detection is not limited to such techniques,and/or western blotting, dot blotting, FACS analyses, and/or the likemay also be used.

In one exemplary ELISA, antibodies are immobilized onto a selectedsurface exhibiting protein affinity, such as a well in a polystyrenemicrotiter plate. Then, a test composition suspected of containing theantigen, such as a clinical sample, is added to the wells. After bindingand/or washing to remove non-specifically bound immune complexes, thebound antigen may be detected. Detection is generally achieved by theaddition of another antibody that is linked to a detectable label. Thistype of ELISA is a simple “sandwich ELISA.” Detection may also beachieved by the addition of a second antibody, followed by the additionof a third antibody that has binding affinity for the second antibody,with the third antibody being linked to a detectable label. The ELISAmay be based on differential binding of an antibody to a protein withArg389 versus Gly389.

In another exemplary ELISA, the samples suspected of containing theantigen are immobilized onto the well surface and/or then contacted withantibodies. After binding and/or washing to remove non-specificallybound immune complexes, the bound anti-antibodies are detected. Wherethe initial antibodies are linked to a detectable label, the immunecomplexes may be detected directly. Again, the immune complexes may bedetected using a second antibody that has binding affinity for the firstantibody, with the second antibody being linked to a detectable label.

Another ELISA in which the antigens are immobilized, involves the use ofantibody competition in the detection. In this ELISA, labeled antibodiesagainst an antigen are added to the wells, allowed to bind, and/ordetected by means of their label. The amount of an antigen in an unknownsample is then determined by mixing the sample with the labeledantibodies against the antigen during incubation with coated wells. Thepresence of an antigen in the sample acts to reduce the amount ofantibody against the antigen available for binding to the well and thusreduces the ultimate signal. This is also appropriate for detectingantibodies against an antigen in an unknown sample, where the unlabeledantibodies bind to the antigen-coated wells and also reduces the amountof antigen available to bind the labeled antibodies.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein or solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, and a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or a third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and/or antibodies with solutions such as BSA, bovine gammaglobulin (BGG) or phosphate buffered saline (PBS)/Tween. These addedagents also tend to assist in the reduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. An example of a washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. This may be an enzyme that willgenerate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact orincubate the first and second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea, or bromocresolpurple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS),or H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generated, e.g., usinga visible spectra spectrophotometer.

ii. Immunohistochemistry

The antibodies of the present invention may also be used in conjunctionwith both fresh-frozen and/or formalin-fixed, paraffin-embedded tissueblocks prepared for study by immunohistochemistry (IHC). For example,immunohistochemistry may be utilized to characterize Fortilin or toevaluate the amount Fortilin in a cell. The method of preparing tissueblocks from these particulate specimens has been successfully used inprevious IHC studies of various prognostic factors, and/or is well knownto those of skill in the art (Brown et al., 1990; Abbondanzo et al.,1990; Allred et al., 1990).

Briefly, frozen-sections may be prepared by rehydrating 50 mg of frozen“pulverized” tissue at room temperature in phosphate buffered saline(PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and/or pelleting again by centrifugation;snap-freezing in −70° C. isopentane; cutting the plastic capsule and/orremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and/or cutting 25-50 serial sections.

Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 hours fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and/or embedding the block in paraffin; and/or cutting upto 50 serial permanent sections.

IV. THERAPY

Once the genotype or the protein sequence of an endothelin gene systemmember of an individual is determined, a therapeutic course of treatmentmay be individualized. In one embodiment of the method, the trait ofinterest is a clinical response exhibited by a patient to sometherapeutic treatment, for example, response to a β-adrenergic receptortargeting agent. As used herein the term “clinical response” means anyor all of the following: a quantitative measure of the efficacy orpotency of the therapy and adverse events (i.e., side effects).

Thus, for example, individuals that are homozygous for an adenosine atnucleotide position +356 in intron 4 of EDN1 (rs2071942) having amedical condition can be placed on a therapy that includes aβ-adrenergic receptor targeting agent such as but not limited tobucindolol. The β-adrenergic receptor targeting agent may beadministered alone or in combination with at least one other agent, suchas a stabilizing compound. The β-adrenergic receptor targeting agent mayalso be administered in combination with a medical device that wouldhave previously been contraindicated by the disease state that requiredthe device.

A. Routes of Administration

Administration of a β-adrenergic receptor targeting agent may be by anynumber of routes including, but not limited to oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, intradermal, intratracheal, intravesicle, intraocular,transdermal, subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, or rectal. Further details on techniques forformulation and administration may be found in the latest edition ofRemington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).In certain embodiments, a β-adrenergic receptor targeting agent isformulated for oral administration.

B. Formulations

Where clinical applications are contemplated, pharmaceuticalcompositions will be prepared in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector or cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrase“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce adverse, allergic, orother untoward reactions when administered to an animal or a human. Asused herein, “pharmaceutically acceptable carrier” includes solvents,buffers, solutions, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the likeacceptable for use in formulating pharmaceuticals, such aspharmaceuticals suitable for administration to humans. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredients of the present invention, itsuse in therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions, providedthey do not inactivate the vectors or cells of the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention may be via any common route so longas the target tissue is available via that route. This includes oral,nasal, or buccal. Alternatively, administration may be by intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection,or by direct injection into cardiac tissue. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions, asdescribed supra.

The active compounds may also be administered parenterally orintraperitoneally. By way of illustration, solutions of the activecompounds as free base or pharmacologically acceptable salts can beprepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations generallycontain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include, forexample, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. Generally, these preparations are sterile and fluid to theextent that easy injectability exists. Preparations should be stableunder the conditions of manufacture and storage and should be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. Appropriate solvents or dispersion media may contain, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

For oral administration the polypeptides of the present inventiongenerally may be incorporated with excipients and used in the form ofnon-ingestible mouthwashes and dentifrices. A mouthwash may be preparedincorporating the active ingredient in the required amount in anappropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan antiseptic wash containing sodium borate, glycerin and potassiumbicarbonate. The active ingredient may also be dispersed in dentifrices,including: gels, pastes, powders and slurries. The active ingredient maybe added in a therapeutically effective amount to a paste dentifricethat may include water, binders, abrasives, flavoring agents, foamingagents, and humectants.

The compositions of the present invention generally may be formulated ina neutral or salt form. Pharmaceutically-acceptable salts include, forexample, acid addition salts (formed with the free amino groups of theprotein) derived from inorganic acids (e.g., hydrochloric or phosphoricacids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic,and the like. Salts formed with the free carboxyl groups of the proteincan also be derived from inorganic bases (e.g., sodium, potassium,ammonium, calcium, or ferric hydroxides) or from organic bases (e.g.,isopropylamine, trimethylamine, histidine, procaine and the like.

Upon formulation, solutions are preferably administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations may easily be administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution generally is suitably buffered andthe liquid diluent first rendered isotonic for example with sufficientsaline or glucose. Such aqueous solutions may be used, for example, forintravenous, intramuscular, subcutaneous and intraperitonealadministration. Preferably, sterile aqueous media are employed as isknown to those of skill in the art, particularly in light of the presentdisclosure. By way of illustration, a single dose may be dissolved in 1ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

1. Controlled/Extended/Sustained/Prolonged Release Administration

Another aspect of this invention provides methods of treating heartfailure patients by delivering the β-adrenergic receptor targeting agentto a patient as a controlled release formulation. As used herein, theterms “controlled,” “extended,” “sustained,” or “prolonged” release ofthe composition of the present invention will collectively be referredto herein as “controlled release,” and includes continuous ordiscontinuous, and linear or non-linear release of the composition ofthe present invention. There are many advantages for a controlledrelease formulation of β-adrenergic receptor targeting agents.

a. Tablets

A controlled release tablet suitable for purposes of this invention isdisclosed in U.S. Pat. No. 5,126,145, which is incorporated by referenceherein. This tablet comprises, in admixture, about 5-30% high viscosityhydroxypropyl methyl cellulose, about 2-15% of a water-solublepharmaceutical binder, about 2-20% of a hydrophobic component such as awaxy material, e.g., a fatty acid, and about 30-90% active ingredient.

b. Films

This invention further provides a prophylaxis for or method of treatinga patient following an invasive cardiac procedure comprisingadministering biodegradable, biocompatible polymeric film comprising aβ-adrenergic receptor targeting agent, such as bucindolol, to a patient.The polymeric films are thin compared to their length and breadth. Thefilms typically have a uniform selected thickness between about 60micrometers and about 5 mm. Films of between about 600 micrometers and 1mm and between about 1 mm and about 5 mm thick, as well as films betweenabout 60 micrometers and about 1000 micrometers, and between about 60and about 300 micrometers are useful in the manufacture of therapeuticimplants for insertion into a patient's body. The films can beadministered to the patient in a manner similar to methods used inadhesion surgeries. For example, a β-adrenergic receptor targetingagent, such as bucindolol, film formulation can be sprayed or droppedonto a cardiac tissue site or artery during surgery, or a formed filmcan be placed over the selected tissue site. In an alternativeembodiment, the film can be used as controlled release coating on amedical device such as a stent, as is discussed in further detail below.

Either biodegradable or nonbiodegradable polymers may be used tofabricate implants in which the β-adrenergic receptor targeting agent isuniformly distributed throughout the polymer matrix. A number ofsuitable biodegradable polymers for use in making the biodegradablefilms of this invention are known to the art, including polyanhydridesand aliphatic polyesters, preferably polylactic acid (PLA), polyglycolicacid (PGA) and mixtures and copolymers thereof, more preferably 50:50copolymers of PLA:PGA and most preferably 75:25 copolymers of PLA:PGA.Single enantiomers of PLA may also be used, preferably L-PLA, eitheralone or in combination with PGA. Polycarbonates, polyfumarates andcaprolactones may also be used to make the implants of this invention.

The amount of the β-adrenergic receptor targeting agent, such asbucindolol, to be incorporated into the polymeric films of thisinvention is an amount effective to show a measurable effect in treatingdiseases having similar pathophysiological states, such as but notlimited to, heart failure, cardiac arrhythmias, hypertension andcardiomyopathy. The composition of the present invention can beincorporated into the film by various techniques such as by solutionmethods, suspension methods, or melt pressing.

c. Transdermal Patch Device

Transdermal delivery involves delivery of a therapeutic agent throughthe skin for distribution within the body by circulation of the blood.Transdermal delivery can be compared to continuous, controlledintravenous delivery of a drug using the skin as a port of entry insteadof an intravenous needle. The therapeutic agent passes through the outerlayers of the skin, diffuses into the capillaries or tiny blood vesselsin the skin and then is transported into the main circulatory system.

Transdermal patch devices which provide a controlled, continuousadministration of a therapeutic agent through the skin are well known inthe art. Such devices, for example, are disclosed in U.S. Pat. Nos.4,627,429; 4,784,857; 5,662,925; 5,788,983; and 6,113,940, which are allincorporated herein by reference. Characteristically, these devicescontain a drug impermeable backing layer which defines the outer surfaceof the device and a permeable skin attaching membrane, such as anadhesive layer, sealed to the barrier layer in such a way as to create areservoir between them in which the therapeutic agent is placed. In oneembodiment of the present invention a formulation of the β-adrenergicreceptor targeting agent is introduced into the reservoir of atransdermal patch and used by a patient who is homozygous for anadenosine at nucleotide position +356 in intron 4 of EDN1 (rs2071942).

d. Medical Devices

Another embodiment contemplates the incorporation of a β-adrenergicreceptor targeting agent, such as bucindolol, into a medical device thatis then positioned to a desired target location within the body,whereupon the β-adrenergic receptor targeting agentelutes from themedical device. As used herein, “medical device” refers to a device thatis introduced temporarily or permanently into a mammal for theprophylaxis or therapy of a medical condition. These devices include anythat are introduced subcutaneously, percutaneously or surgically to restwithin an organ, tissue or lumen. Medical devices include, but are notlimited to, stents, synthetic grafts, artificial heart valves,artificial hearts and fixtures to connect the prosthetic organ to thevascular circulation, venous valves, abdominal aortic aneurysm (AAA)grafts, inferior venal caval filters, catheters including permanent druginfusion catheters, embolic coils, embolic materials used in vascularembolization (e.g., PVA foams), mesh repair materials, a Dracon vascularparticle orthopedic metallic plates, rods and screws and vascularsutures.

In one embodiment, the medical device such as a stent or graft is coatedwith a matrix. The matrix used to coat the stent or graft according tothis invention may be prepared from a variety of materials. A primaryrequirement for the matrix is that it be sufficiently elastic andflexible to remain unruptured on the exposed surfaces of the stent orsynthetic graft.

C. Dosages

The amount of a β-adrenergic receptor targeting agent that isadministered or prescribed to the patient can be about, at least about,or at most about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400,410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500 mg, or any rangederivable therein. Alternatively, the amount administered or prescribedmay be about, at least about, or at most about 0.001, 0.002, 0.003,0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05,0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7.3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 mg/kg,or any range derivable therein, with respect to the weight of thepatient.

When provided in a discrete amount, each intake of a β-adrenergicreceptor targeting agent can be considered a “dose.” A medicalpractitioner may prescribe or administer multiple doses of bucindololover a particular time course (treatment regimen) or indefinitely.

A β-adrenergic receptor targeting agent may be administered 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50,60, 70, 80, or more times or any range derivable therein. It is furthercontemplated that the drug may be taken for an indefinite period of timeor for as long as the patient exhibits symptoms of the medical conditionfor which a β-adrenergic receptor targeting agent was prescribed oradministered. Also, the drug may be administered every 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24hours, or 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5 weeks, or 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, or any range derivabletherein. Alternatively, it may be administered systemically over anysuch period of time and be extended beyond more than a year.

D. Other Therapeutic Options

In another embodiment, it is envisioned to use a β-adrenergic receptortargeting agent in combination with other therapeutic modalities. Thus,in addition to the therapies described above, one may also provide tothe patient more “standard” pharmaceutical cardiac therapies. Examplesof other therapies include, without limitation, other beta blockers,anti-hypertensives, cardiotonics, anti-thrombotics, vasodilators,hormone antagonists, inotropes, diuretics, endothelin antagonists,calcium channel blockers, phosphodiesterase inhibitors, ACE inhibitors,angiotensin type 2 antagonists and cytokine blockers/inhibitors, andHDAC inhibitors.

Combinations may be achieved by contacting cardiac cells with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the agent. Alternatively, the therapyusing a β-adrenergic receptor targeting agent may precede or followadministration of the other agent(s) by intervals ranging from minutesto weeks. In embodiments where the other agent and expression constructare applied separately to the cell, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the agent and expression construct would still beable to exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one would typically contact the cellwith both modalities within about 12-24 hours of each other and, morepreferably, within about 6-12 hours of each other, with a delay time ofonly about 12 hours being most preferred. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

It also is conceivable that more than one administration of eitherβ-adrenergic receptor targeting agent or the other agent will bedesired. In this regard, various combinations may be employed. By way ofillustration, where the β-adrenergic receptor targeting agent is “A” andthe other agent is “B”, the following permutations based on 3 and 4total administrations are exemplary:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/BOther combinations are likewise contemplated.

V. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Endothelin Gene System Member Analysis—Methodology

Clinical Studies.

309 subjects with idiopathic dilated cardiomyopathy were studied fromthe Beta-Blocker Evaluation of Survival Trial (BEST) (2001). BEST was amulticenter, randomized, prospective trial of patients with NYHA classIII or IV heart failure and an ejection fraction <35% comparingmortality in adults treated with placebo or the non-selective β-blockerbucindolol. The BEST DNA bank, initiated in the study's second year,collected samples from 38% of the original participants. The inventorsrequested samples from idiopathic dilated cases, rather than the entirecohort, to ensure a more homogenous heart failure population. Inparticular, the inventors sought to avoid studying genetic ischemicforms of heart failure where acquired macrovascular changes in arterialstiffness or tone could potentially mask or override inherentmicrovascular phenotypes mediated by variation in endothelin genes.Written informed consent was obtained from each participant and all DNAwas coded to maintain confidentiality. The genotyping project wasapproved by the BEST DNA Bank Oversight Committee and by the ColoradoMultiple Institutional Review Board

Selection of EGS Polymorphisms.

EGS SNPs were selected prior to the release of HapMap data using anextensive literature review and a search of the NCBI SNP database (dbSNPbuild 108). The six EGS genes studied included three endothelin genes(EDN1, EDN2, EDN3), both endothelin receptors A and B (EDNRA, EDNRB) andthe endothelin converting enzyme 1 (ECE1). SNPs predicting changes inamino acid sequence of EGS proteins were prioritized for study. SNPs innon-coding regions (intronic and promoter regions) and synonymous SNPswere also studied when prior reports suggested a positive associationwith a phenotype in humans and/or a functional effect had beendemonstrated in an in vitro model. Twenty-two EGS SNPs, includingfifteen nonsynonymous SNPs, were identified by searching publishedliterature and the dbSNP database (Table 1) (Herrmann et al., 2001;Charron et al., 1999; Vasku et al., 2002). At the start of the study,population frequencies of thirteen EGS SNPs were not reported in dbSNPand these SNPs proved rare in the population and were not analyzedfurther (rs#: 5798, 5799, 457755, 457651, 5345, 5346, 1801710, 2228271,5347, 5350, 5352, 3026902, 3026906). Nine SNPs in four genes with minorallele frequencies of 5% or greater and were included in the statisticalanalysis. In silico analyses of EGS SNPs for predicted effects onprotein secondary structure used GOR IV and PSIPRED algorithms (Garnieret al., 1996; Jones, 1999); possible effects on splicing used in theGeneSplicer program (Pertea et al., 2001).

TABLE 1 Endothelin Gene System Polymorphisms Initially Selected forStudy rs BEST MAF Gene number SNP Protein Location NCBI MAF (n = 309)EDN1 1800997 −138 DelA No change 5′ UTR 0.19 0.25 5369 G/A Glu106 Exon 30.11 0.12 2071942 G/A No change IVS-4 0.16 0.25 5370 G/T Lys198Asn Exon5 0.26 0.24 EDNRA 1801708 A/G No change 5′ UTR 0.49 0.44 5333 C/T His323Exon 6 0.31 0.34 5343 C/T No change 3′ UTR 0.25 0.34 EDNRB 5351 A/GLeu277 Exon 5 0.43 0.41 ECE1 1076669 C/T Thr341Ile Exon 9 0.04 0.07SNP-single nucleotide polymorphism; UTR-untranslated region;IVS-intervening sequence (intron); allele frequency for rs1800997[Charron et al., 1999]; MAF-Minor allele frequency listed as reported inNCBI dbSNP (build 127). BEST-Beta Blocker Evaluation of Survival Trial;NR = not reported

Genotyping.

Genotyping was performed by the University of Colorado CardiovascularInstitute laboratory using a MSQ96 MA Pyrosequencer (Biotage, Uppsala,Sweden) and primers were designed using software provided by themanufacturer. PCR reactions were first performed with primer pairsflanking each SNP; one of each primer pair also contained an M13sequence (5′,5′-Biotin-CAGGAAACAGCTATGAC-3′) (SEQ ID NO:1) to allow forincorporation of a biotinylated ‘universal’ primer containing the sameM13 sequence. Pyrosequencing was then performed using sequencing primersdesigned specifically for each SNP.

For rs5370, the assay used the following primers:forward-CTTCTTTTGCCAAAGGGTGA (SEQ ID NO:2),reverse-biotin-AGGGTGGAGAGTGCAGAGTC (SEQ ID NO:3),sequencing-CCAAGCTGAAAGGCA (SEQ ID NO:4). For rs2071942 the assay usedthe following primers:forward-biotin-CAGGAAACAGCTATGACCAGCCTTTGCCTCTCTGAGTC (SEQ ID NO:5),reverse-GGCCATCTGAATAACTGCAAC (SEQ ID NO:6) sequencing-GCTCCCCAAAATGAT(SEQ ID NO:7). Results for selected SNPs underwent repeat genotyping ofa random subset of 10% of the samples for validation and qualitycontrol.

Statistical Analysis.

Time to each of two endpoints was examined: all cause death and acombined endpoint comprised of first heart failure hospitalization orall cause death, using the Cox proportional hazards model. Genotype wastreated a priori as a continuous variable, consistent with an alleledosage effect. Analyses were conducted using R and SAS. Primary analysesconsisted of separate tests of a main genotype effect on time to eventfor each SNP within each treatment arm: placebo (N=159) and bucindolol(N=150). Secondary analyses, in the combined sample (N=309), consistedof tests of a genotype-treatment pharmacogenetic interaction and of amain genotype effect independent of treatment arm. Secondary analyses,comprised of interaction tests and estimation of within-genotypebucindolol:placebo hazard ratios, were also performed. Ethnicity was notincluded as a factor in initial Cox models, because of the relativelysmall sample. However, post-hoc analyses (within ethnic group, andincluding ethnicity-genotype interactions) were used to explore whetherinitial results were due to unaccounted for ethnic stratification.Linkage disequilibrium was assessed by taking the R² value (squaredcorrelation) of the genotypes (coded as 0, 1 or 2) between two SNPs.

The inventors had two a priori reasons for emphasizing within-treatmentarm effects, over the more usual interaction test. As a gene-treatmentinteraction effect cannot exist without a main genetic effect in atleast one of the treatment groups, the first test investigated whetherthere was any such main genetic effect before testing whether thateffect differed in a way that depended on treatment (i.e., apharmacogenetic interaction). Second, relative power with approximatelyequal sample sizes was examined, assuming a possible pharmacogeneticeffect with no or a negligible effect of genotype in one group, say theplacebo group, and a more substantial effect in the other, active-drug,group. In that case, the values of the active-drug genotype effect andthe interaction effect (written as the difference of within-treatmenteffects) are the same. The standard error of the interaction effect isapproximately √2×S, where S is the standard error of thewithin-treatment effect. Thus, the most powerful test for any type ofgenotype effect in this setting is the within-treatment test in the armwith a genetic effect. This is generally true, unless the geneticeffects work in opposite directions in the two treatment arms. The testfor a main genotype effect, independent of treatment group, would be themost powerful in the absence of a pharmacogenetic effect.

To account for the large number of simultaneous tests, adjusted p-valuesobtained from a multiple-testing permutation procedure are reported(Westfall and Young, 1993). Based on the actual data set, 5000 randomdata sets were generated by randomly permuting the time to event datarelative to the genotype data, separately for patients on placebo andfor patients on bucindolol. Adjusted p-values for all within treatmentarm tests are based on 36 comparable tests (2 outcomes×2 treatmentarms×9 SNPs). Adjusted p-values for interaction tests are based on 18tests (2 outcomes×9 SNPs).

Cox model analysis was also used to examine a variety of morecomplicated models, and to describe the quantify the effects ofbucindolol in terms of bucindolol:placebo hazard ratios, withingenotype. Kaplan-Meier plots and log-rank tests were also examined. Allp-values were based on two-sided tests. The statistical analysis wasperformed at the Veterans Affairs Cooperative Studies Program DNA BankCoordinating Center.

Example 2 Demographic and Clinical Characteristics of Test Groups

The placebo and bucindolol groups were similar with respect todemographic and clinical characteristics (Table 2). There were nosignificant differences between the placebo and bucindolol groups withrespect to baseline heart rate, systolic blood pressure, body massindex, or baseline cardiovascular medications. Genotype frequencies atall nine EGS SNPs were consistent with prior reports (Table 1). All butone SNP were in Hardy-Weinberg equilibrium; disequilibrium at EDNRA A/G(5′UTR) appeared to be generated by ethnic stratification, sincegenotypes were in Hardy-Weinberg equilibrium when analyzed within eachethnic group.

TABLE 2 Comparison of treatment groups Placebo Bucindolol Variable (N =159) (N = 150) p-value* Median Age (years) 57.0 59.0 0.50 Gender Male106 (67%) 107 (71%) 0.38 Female 53 (33%) 43 (29%) Race NonBlack 128(81%) 112 (75%) 0.22 Black, non-Hispanic 31 (19%) 38 (25%) NHYA ClassClass III 151 (95%) 141 (94%) 0.71 Class IV 8 (5%) 9 (6%) Medianduration of HF 24.0 26.5 0.94 (months) Diabetes 39 (25%) 50 (33%) 0.09Hypertension 72 (45%) 76 (51%) 0.34 Current Tobacco Use 23 (14%) 20(13%) 0.77 Mean Arterial Pressure 87.4 (11.8) 88.2 (12.3) 0.56 (SD) MeanLVEF (SD) 24.5 (7.0) 24.5 (7.0) 0.93 Mean Serum creatinine 1.1 (0.3) 1.2(0.3) 0.08 mg/dL (SD) Mean Plasma 460.5 (311) 468.3 (253) 0.83Norepinephrine pg/ml (SD)

Example 3 Identification of Two SNPs with Beneficial Treatment Effects

After controlling for multiple testing, no EGS variant had a significanteffect on time to all cause death in either treatment arm, or on time tothe combined outcome in patients on placebo. However, two SNPs in EDN1(G/T Lys198Asn and G/A (IVS-4)) had highly significant genotype effectson time to the combined endpoint of first heart failure hospitalizationor all cause death for patients on bucindolol, even after rigorousadjustment for multiple tests (FIG. 1, Table 3). The alleles in the morecommon G-Lys haplotype were each associated with a better outcome. Thispharmacogenetic interaction resulted in an apparent beneficial effect ofbucindolol in the common homozygotes, a harmful effect in the rarehomozygotes and a neutral effect in the heterozygotes (Table 4). Onlythe beneficial effect in the common homozygote group, which had thelargest numbers of subjects and events, was statistically significant.Hospitalization was not considered a primary or secondary endpoint inBEST and is not included in the reported analyses. Results forhospitalization are similar to those for the combined endpoint. However,the inventors felt including another post hoc analysis would not add tothe results. When hospitalization alone is considered, excluding deathswithout prior hospitalization appears to decrease precision, increasingthe standard error by about 10%.

G/A (IVS-4) and Lys198Asn were in tight linkage disequilibrium (genotypeR²=0.85), appearing primarily in unphased genotypes consistent with theG-Lys and A-Asn haplotypes. Thus, it was not possible to distinguishstatistically between the SNPs relative importance in a two-SNP model.Neither SNP was independently statistically significant in the presenceof the other, although the null model with neither SNP was clearlyrejected. EDN1 G/A (IVS-4) and Lys198Asn also had significanttreatment-genotype interactions (Table 3).

Two other SNPs, EDN1 Glu106 (p=0.04) and ECE1 Thr341Ile (p=0.03) hadsignificant main effects for the combined outcome, but were notsignificant after adjustment for multiple tests. EDNRA His323 (p=0.03)had a significant treatment-genotype interaction for time to all-causedeath, before adjustment for multiple tests. The test for a maingenotype effect in the combined data set yielded no significant resultsfor either outcome.

TABLE 3 Effects of One-Allele Changes in the Allele Dosage Cox ModelTreatment SNP Group N E MAF HR 95% CI p adj p* Bucindolol G/A (IVS-4)All 150 49 25% 2.2 1.4-3.3 <0.001 0.01 Non-Hispanic Whites 100 27 24%1.9 1.09-3.45 0.02 Non-Hispanic Blacks 38 16 22% 1.58 0.67-3.76 0.30Lys198Asn All 150 49 24% 2.0 1.3-3.0 0.001 0.03 Non-Hispanic Whites 10027 23% 1.9 1.05-3.30 0.03 Non-Hispanic Blacks 38 16 22% 1.5 0.75-3.100.25 Placebo G/A (IVS-4) All 159 81 25% 0.83 0.5-1.3 0.41 1.00Non-Hispanic Whites 112 41 25% 0.9 0.55-1.60 0.8 Non-Hispanic Blacks 3116 26% 0.5 0.21-1.20 0.12 Lys198Asn All 158 60 25% 0.9 0.6-1.3 0.48 1.00Non-Hispanic Whites 111 40 24% 0.9 0.54-1.60 0.78 Non-Hispanic Blacks 3116 27% 0.5 0.22-1.21 0.13 Data shown are for combined endpoint (firstheart failure hospitalization or all-cause mortality); HR-hazard ratiosassociated with each copy of allele A or Asn, respectively, under anallele dosage model. For bucindolol by SNP interaction tests, p values(adjusted p values**) were 0.002 (0.03) and 0.006 (0.07) for IVS-4 andLys198Asn, respectively. A non-significant trend for interaction wasobserved for both SNPs for both ethnic subgroups. *adjusted p value for36 tests of genotype within treatment arm in primary analysis.**Adjusted p values for 18 interaction tests. Post-hoc analyses byethnic sub-group were not included in p value adjustments. N-number ofsubjects (All includes subjects from other ethnicities); E-number ofevents; MAF-minor allele frequency in sample

TABLE 4 Hazard Ratios for Bucindolol Versus Placebo by EDN1 Genotype SNPGroup N* E HR 95% CI p value G/A (IVS-4) GG 171 56 0.5 0.3-0.9 0.01 GA121 44 1.1 0.6-1.9 0.85 AA 17 10 3.2  0.8-12.5 0.10 Lys198Asn Lys/Lys176 58 0.5 0.3-0.9 0.02 Lys/Asn 115 41 1.1 0.6-2.0 0.77 Asn/Asn 17 102.5 0.6-9.9 0.19 Data shown are for combined endpoint (first heartfailure hospitalization or all cause mortality); N-number of subjects;E-number of events; HR-hazard ratio; 95% CI-95% confidence interval;*One individual was not successfully genotyped at Lys198Asn

Example 4 Secondary Analyses: Ethnic Study

Since Blacks were previously reported to experience a worse response inBEST, the inventors conducted secondary analyses in the two largestethnic subgroups: non-Hispanic Blacks and non-Hispanic Whites. Both G/A(IVS-4) and Lys198Asn showed significant effects for the combinedoutcome in non-Hispanic Whites in the bucindolol treated group; inBlacks where the sample size was much smaller, the hazard ratios wereconsistent, but no longer significant. In each group, there was anon-significant trend for a treatment-SNP interaction for both G/A(IVS-4) and Lys198Asn. ECE1 Thr341lle could not be tested innon-Hispanic Blacks, which included a single carrier of the minorallele. Its significance in bucindolol treated subjects increased whenthe analysis was restricted to non-Hispanic Whites (p=0.008). EDN1Glu106 was significant in non-Hispanic Whites (p=0.03), but not innon-Hispanic Blacks.

Example 5 Two-SNP Models Including Either EDN1 G/A (IVS-4) or Lys198Asn

All possible two-SNP models were tested, including either EDN1 G/A(IVS-4) or Lys198Asn and one of the remaining seven SNPs, both with andwithout a SNP-SNP interaction term, in post-hoc analyses. ECE1 Thr341Ilewas significant in the two-SNP models including either EDN1 G/A (IVS-4)(p=0.04) or Lys198Asn (p=0.04), but only in the no-interaction models.Thus, ECE1 Thr341Ile might contribute an effect on the combined outcomeindependent of the EDN1 SNPs. In the EDN1 Lys198Asn-ECE1 Thr341Ilemodel, the hazard ratios (HRs) are 1.97 (95% CI 1.31, 2.96) and 1.81(95% CI 1.04, 3.15), respectively. In the EDN1 G/A (IVS-4)-ECE1Thr341Ile model, the HRs are 2.09 (95% CI 1.37, 3.20) and 1.78 (95% CI1.02, 3.12), respectively. Thus, the effect of the ECE1 SNP is estimatedto be almost as strong as those of the two EDN1 variants. The weakerstatistical significance for ECE1 Thr341Ile may be due to its lessbalanced allele frequencies, which reduce power. In contrast, EDN1Glu106 was no longer significant when added to a model including eitherG/A (IVS-4) or Lys198Asn. Thus, the significance of EDN1 Glu106 in aone-SNP model might merely reflect its highly significant linkagedisequilibrium with those SNPs (p<0.001).

Results were qualitatively unchanged in other post hoc analyses. Liggettet al. (2006) previously genotyped reduced all-cause mortality inbucindolol treated subjects carrying the Arg-389 allele of thebeta-1-adrenergic receptor (ADRB) in the 1040 BEST DNA Bank subjects.This sample represents a subset of that earlier study. The inventorsobtained these genotypes for the subjects and fit two-SNP models withone EDNS SNP and the ADRB Gly389Arg variant as described in the previousparagraph. The inventor's estimates and significance results werequalitatively unchanged.

For all SNPs that were significant under the allele-dosage model used inprimary analyses, neither a dominant nor a recessive genetic model fitbetter based on the value of the log-likelihood. In some of these cases,either the dominant or recessive model failed to detect significance. Nonon-significant SNP in the primary analyses became significant under arecessive of dominant model. Post hoc adjustment for possiblyconfounding effects of medications did not qualitatively affect primarySNP results. A secondary logistic regression analysis of genetic effectson adverse events found no significant results, when adjusted formultiple tests.

The foregoing description is considered as illustrative only of theprinciples of the invention. Further, since numerous modifications andchanges will readily occur to those skilled in the art, it is notdesired to limit the invention to the exact construction and process asdescribed above. Accordingly, all suitable modifications and equivalentsmay be resorted to falling within the scope of the invention as definedby the claims that follow.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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The invention claimed is:
 1. A method for treatment of a human patientwith bucindolol comprising administering to the patient at least 50 mgof bucindolol formulated for oral administration after the patient isdetermined to be homozygous for guanine at position +356 in intron 4 ofEDN1 (rs2071942) or homozygous for lysine at amino acid 198 of the EDN1protein.
 2. The method of claim 1, wherein the patient is determined tobe homozygous by a method that comprises performing on the EDN1 genefrom the patient pyrosequencing, chain terminating sequencing,restriction digestion, allele-specific polymerase reaction,single-stranded conformational polymorphism analysis, genetic bitanalysis, temperature gradient gel electrophoresis, ligase chainreaction, or microarray hybridization.
 3. The method of claim 1, whereinthe patient is determined to be homozygous for guanine at position +356in intron 4 of EDN1 (rs2071942) or homozygous for lysine at amino acid198 of the EDN1 protein by obtaining a patient history.
 4. The method ofclaim 1, further comprising obtaining a biological sample from thepatient.
 5. The method of claim 1, wherein the patient has symptoms ofor has been diagnosed with a medical condition comprising heart failureor cardiomyopathy.
 6. A method for treating a human patient withbucindolol comprising orally administering at least 50 mg bucindolol tothe patient after the patient has been genotyped at nucleotide position+356 in intron 4 of EDN1 (rs2071942) of the patient and determined to behomozygous for guanine.
 7. The method of claim 6, wherein the patienthas been genotyped by a process comprising pyrosequencing, chainterminating sequencing, restriction digestion, allele-specificpolymerase reaction, single-stranded conformational polymorphismanalysis, genetic bit analysis, temperature gradient gelelectrophoresis, ligase chain reaction, or microarray hybridization. 8.A method for treating a human patient with bucindolol comprising orallyadministering to the patient at least 50 mg of bucindolol after thepatient has been genotyped at nucleotide position +61 in exon 5 of EDN1(rs5370) and determined to be homozygous for a nucleotide encodinglysine.
 9. The method of claim 8, wherein the patient has been genotypedby a process comprising pyrosequencing, chain terminating sequencing,restriction digestion, allele-specific polymerase reaction,single-stranded conformational polymorphism analysis, genetic bitanalysis, temperature gradient gel electrophoresis, ligase chainreaction, or microarray hybridization.